Preparation method and applications of a long-acting antitumor organic and inorganic bone binder

Abstract

This invention discloses a method for preparing and using a long-acting antitumor organic and inorganic bone binder, which is prepared by polymerization, blending, and freeze-drying of porous calcium phosphate and antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate is prepared by controlling its porous structure and composition to achieve degradation, thereby slowly releasing calcium ions, improving osteogenic properties, and achieving high bonding strength with human bone at the postoperative bone tumor defect site. Simultaneously, leveraging the high bonding strength and long-acting antitumor properties of the Tremella fuciformis polysaccharide hydrogel grafted with one or two antioxidants such as ascorbic acid, tea polyphenols, flavonoids, and tocopherols, the porous calcium phosphate and antioxidant-grafted Tremella fuciformis polysaccharide hydrogel are combined to form a covalently cross-linked network structure, further improving the bonding strength of the bone binder, promoting osteogenic properties with host bone tissue, and providing a long-lasting antitumor cycle.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing and using a long-acting anti-tumor organic and inorganic bone binder. Background Technology

[0002] Bone tumors can occur at any age in humans. Osteosarcoma, a life-threatening malignant tumor with an extremely low incidence (only 3%), commonly occurs in adolescents or young adults aged 10-25, and its adjuvant therapy phase requires 2-5 months. Osteochondroma, a benign tumor with a very high incidence (up to 4%), requires 1-4 months of adjuvant therapy. Clinically, bone adhesives are currently mainly used to fill bone defects created after tumor resection in bone tumor treatment. Furthermore, they are often loaded with antitumor drugs such as calmodulin inhibitors and bone resorption inhibitors for adjuvant therapy. Currently, commonly used bone adhesives are classified into organic and inorganic types based on their chemical composition. Organic bone adhesives, such as hydrogel adhesives like gelatin, chitosan, and bioactive glass, and polymethyl methacrylate adhesives, have advantages such as good biocompatibility, bioactivity, and high mechanical strength, but they also have drawbacks such as poor ability to promote osteoblast growth and bone tissue regeneration, and poor interfacial bonding with bone tissue. Inorganic bone binders, such as calcium phosphate binders and calcium sulfate binders, have advantages such as similar chemical composition and structure to bone tissue, promoting bone tissue regeneration and repair, and controllable degradation rate, and also possess certain bonding properties. However, they suffer from disadvantages such as low self-bonding strength and easy disintegration, affecting long-term use. Furthermore, single organic or inorganic bone binders loaded with antitumor drugs exhibit problems in treatment, including poor release of antitumor drugs, concentrations lower than the effective concentration for inhibiting tumor cell growth, and shorter treatment cycles than those for inhibiting tumor cell growth.

[0003] Given the advantages and disadvantages of the aforementioned bone binders, research on organic-inorganic composite bone binders is of great significance. Currently, researchers are obtaining novel composite bone binder materials through physical or chemical modifications such as metal ion doping, ceramic fiber modification, and drug-loaded microspheres. These novel materials possess advantages such as excellent bioactivity, but still suffer from drawbacks such as low bond strength due to poor interaction between the bone binder and human bone, and a short anti-tumor cycle due to poor ability to inhibit tumor cell growth, thus limiting their application in clinical treatment.

[0004] Chinese patent "An Injectable Bone Repair Hydrogel of Bioactive Calcium Phosphate / Fibrin Composite" describes the preparation of an injectable bone repair hydrogel by mixing bioactive calcium phosphate, fibrin, and thrombin. This bone repair hydrogel exhibits good biocompatibility, but suffers from poor tumor cell growth inhibition and a short anti-tumor cycle.

[0005] Chinese patent "A Magnesium-Based Medical Bone Bonding Material and Its Preparation Method" describes a magnesium-based medical bone bonding material prepared by mixing magnesium oxide, hydrogen phosphate, and calcium phosphate as the solid phase and citric acid aqueous solution or sodium citrate aqueous solution as the liquid phase. The composite bone bonding material exhibits a bonding strength of 2–5 MPa and a degradation weight loss rate of 20 ± 5% over four weeks. However, it still suffers from low bonding strength due to the high reactivity of magnesium, which readily reacts with oxygen and water in the body to form compounds such as magnesium oxide or magnesium hydroxide, resulting in weak interaction with human bone. Furthermore, it exhibits a short anti-tumor cycle and cannot effectively inhibit tumor cell growth; prolonged use may lead to recurrence and metastasis of bone tumors, limiting its application in the field of biomedical materials technology.

[0006] Chinese patent "Bisphosphonic Acid-Containing Polyamino Acid Copolymer, Anti-Bone Tumor Bone Material and Its Preparation" describes a process of uniformly mixing and melting a bisphosphonic acid-containing polyamino acid copolymer with a calcium salt compound to obtain a bisphosphonic acid-containing polyamino acid copolymer / calcium salt composite material with anti-bone tumor properties. This composite material exhibits inhibitory effects against chondrosarcoma, osteosarcoma, and Ewing's sarcoma, with a degradation rate of 43.6% within one month. However, it still suffers from drawbacks such as low bonding strength due to the relatively weak interaction between the polymer and bone powder, poor ability to inhibit tumor cell growth, and a short anti-tumor cycle.

[0007] Chinese patent "A Method for Preparing a Photothermal Anti-Bone Tumor Coating on a Magnesium Alloy Surface" describes a method for forming a selenium-doped hydroxyapatite embedded black phosphorus nanosheet@β-TCP nanofiber coating through selenium doping of hydroxyapatite microparticles, synthesis of β-TCP nanofibers, and electrophoretic co-deposition of black phosphorus nanosheets. This composite material exhibits inhibition of tumor cell growth (tumor cell mortality >90% under light irradiation), but suffers from a short anti-tumor cycle and low bonding strength. Long-term use leads to a decrease in drug release concentration, causing tumor cell recurrence, thus limiting its application in the field of biomedical materials technology.

[0008] To address the issues of low bonding strength due to weak interaction between the novel composite bone adhesive material and human bone, as well as poor ability to inhibit tumor cell growth after long-term use, designing a long-acting anti-tumor organic and inorganic bone adhesive is of significant research importance. Summary of the Invention

[0009] This invention addresses the shortcomings of existing composite bone adhesives, such as low bonding strength, inhibition of tumor cell growth, and short anti-tumor cycle. It is prepared by polymerization, blending, and freeze-drying of porous calcium phosphate and antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate is prepared by controlling its porous structure and composition to achieve degradation, thereby slowly releasing calcium ions, improving osteogenic properties, and achieving high bonding strength with human bone at the postoperative bone tumor defect site. Simultaneously, leveraging the high bonding strength and long-lasting anti-tumor properties of Tremella fuciformis polysaccharide hydrogel grafted with one or two antioxidants such as ascorbic acid, tea polyphenols, flavonoids, and tocopherols, the porous calcium phosphate and antioxidant-grafted Tremella fuciformis polysaccharide hydrogel are combined to form a covalently cross-linked network structure, further improving the bonding strength of the bone adhesive, promoting osteogenic properties with host bone tissue, and providing a long-lasting anti-tumor cycle.

[0010] This invention uses porous calcium phosphate as the inorganic material. Firstly, the porous structure of porous calcium phosphate increases the surface area for cell adhesion, thereby improving the bonding strength of the bone adhesive. Secondly, by filling the defect area of ​​postoperative bone tumors, it provides an environment conducive to bone cell growth and differentiation, promoting bone tissue regeneration and repair. Simultaneously, porous calcium phosphate acts as a drug carrier, adsorbing or encapsulating anti-tumor drugs within its porous structure and achieving slow, continuous release to increase drug concentration at the treatment site. This invention uses Tremella fuciformis polysaccharide as the anti-tumor drug. Tremella fuciformis polysaccharide has excellent ability to inhibit tumor cell growth, enhances the body's anti-tumor immune capacity by stimulating the immune system, promotes the activity of macrophages, natural killer cells, and lymphocytes, and enhances the attack effect of immune cells on tumor cells. Alternatively, it inhibits tumor cell metabolism by disrupting telomerase in tumor cells. Furthermore, since oxidative stress is an important factor in tumor occurrence and development, and Tremella fuciformis polysaccharide has antioxidant properties, it can scavenge free radicals in the body and reduce cell damage caused by oxidative stress. Secondly, the Tremella fuciformis polysaccharide hydrogel has high bonding strength (bonding strength 300-500 MPa). Because the molecular structure of Tremella fuciformis polysaccharide contains a large number of stable ester bonds and glycosidic bonds, it has the advantage of a long degradation cycle. Combining the advantages of porous calcium phosphate and Tremella fuciformis polysaccharide, this invention combines the two through polymerization and blending to construct a long-acting anti-tumor organic and inorganic bone binder.

[0011] The present invention provides a method for preparing a long-acting antitumor organic and inorganic bone binder, comprising the following steps:

[0012] Step 1, Preparation of porous calcium phosphate: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate is reacted with anhydrous calcium chloride to obtain calcium phosphate precipitates with different calcium-to-phosphorus ratios. After standing and filtration, the precipitates are dried to obtain calcium phosphate powder. Polyvinyl alcohol and polyacrylic acid are dissolved in deionized water to obtain solution A as a pore-forming agent. The calcium phosphate powder is dispersed in anhydrous ethanol and solution A is added and ultrasonicated. After completion, the powder is allowed to stand and the supernatant is removed. The powder is then washed by centrifugation with deionized water, dried, and sieved to obtain porous calcium phosphate powder.

[0013] Step 2, Preparation of antioxidant-grafted Tremella polysaccharide hydrogel by free radical polymerization: First, add 1-8g of Tremella polysaccharide to 100-800mL of deionized water to obtain a Tremella polysaccharide solution; simultaneously, add hydrochloric acid solution dropwise, and then heat in a water bath at 40-50℃ for 15-30min to dissolve, obtaining a Tremella polysaccharide gel; second, add an antioxidant to graft the Tremella polysaccharide gel to obtain a mixture; stir the mixture to obtain an antioxidant-grafted Tremella polysaccharide gel.

[0014] Step 3, Preparation of porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogel: First, one of the following dispersants—polyvinylpyrrolidone, polyacrylic acid, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate—and the crosslinking agent—methacrylamide or dimethacrylamide—are dissolved in 80–120 mL of deionized water by stirring for 5–15 min to obtain dispersant solution and crosslinking agent solution, respectively; the initiator is dissolved in 40–60 mL of anhydrous ethanol by stirring for 5–15 min to obtain initiator solution; second, the... The obtained porous calcium phosphate powder is uniformly dispersed in a dispersant solution to obtain a first solution; subsequently, the first solution is heated and stirred with a crosslinking agent solution, an initiator solution, and the antioxidant-grafted Tremella fuciformis polysaccharide hydrogel obtained in step 3 to obtain porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel; porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel is prepared by freeze-drying to obtain dried porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which is then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0015] Preferably, in step 1, the mass ratio of disodium hydrogen phosphate dodecahydrate to anhydrous calcium chloride is in the range of 1.5:1 to 1.8:1, the drying temperature is 30 to 60°C, and the drying time is 4 to 6 hours.

[0016] The pore-forming agent selected in step 1 is polyvinyl alcohol or polyacrylic acid, with a mass fraction of 40%, 50%, 60%, or 70%.

[0017] In step 1, calcium phosphate powder is ultrasonically dispersed uniformly in anhydrous ethanol and added to solution A for further ultrasonication. The mass of calcium phosphate powder is 0.5-1.5g, and the ultrasonication lasts for 5-20 minutes. After adding solution A, the ultrasonication continues for 15-30 minutes.

[0018] In step 1, during the standing, centrifugation, washing and drying process, the plant is left to stand at room temperature for 12 to 24 hours, centrifuged and washed 3 to 6 times, dried at 80 to 120°C, and dried for 12 to 24 hours.

[0019] The porous calcium phosphate powder obtained in step 1 has a pore size range of 10–100 μm and a porosity range of 40%–80%.

[0020] Preferably, in step 2, the pH of the Tremella polysaccharide solution is adjusted to 3-6 using hydrochloric acid;

[0021] The antioxidants selected in step 2 are one or two of ascorbic acid, tea polyphenols, flavonoids, and tocopherols, and the mass fraction of the antioxidants is 5% or 10%.

[0022] In step 2, the antioxidant-grafted Tremella polysaccharide gel is prepared by heating in a water bath at 40–50°C for 15–30 minutes.

[0023] Preferably, the dispersant in step 3 is one of polyvinylpyrrolidone, polyacrylic acid, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate, with a mass fraction ranging from 3% to 5%; the crosslinking agent is methacrylamide or dimethacrylamide, with a mass fraction ranging from 6% to 8%; and the initiator is 2,2'-azobisisobutyronitrile or ammonium persulfate, with a mass fraction ranging from 1% to 4%.

[0024] The mass of porous calcium phosphate powder in step 3 is 1-2g;

[0025] In step 3, the first solution, the crosslinking agent solution, and the initiator solution are heated to a temperature of 65–75°C for 2–4 hours.

[0026] In step 3, porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogels are prepared by freeze-drying. The freezing temperature is -50℃ to -80℃, the vacuum degree is 10 to 30 Pa, and the drying time is 12h to 36h.

[0027] The use of a long-acting anti-tumor organic and inorganic bone binder: The binder obtained by the preparation method of a long-acting anti-tumor organic and inorganic bone binder is used in the field of biomedical materials technology.

[0028] Compared with the prior art, the beneficial effects of the present invention are:

[0029] Firstly, this invention uses a pore-forming agent to prepare porous calcium phosphate with a pore size ranging from 10 to 100 μm and a porosity ranging from 40% to 80%. The porous calcium phosphate permeates into the pores through body fluids and reacts with calcium phosphate compounds on the material surface to form soluble calcium and phosphate ions. Subsequently, it recrystallizes in the body fluids, forming new calcium phosphate deposits. These deposits fill the pores of the porous calcium phosphate and contact and bond with the bone tissue surrounding the postoperative bone tumor defect. Ultimately, the porous calcium phosphate is completely degraded and replaced by newly formed bone tissue. Simultaneously, the degradation of the porous calcium phosphate slowly releases calcium ions, improving osteogenic properties. Furthermore, by leveraging the anti-tumor properties of Tremella fuciformis polysaccharide, high bonding strength is achieved at the postoperative bone tumor defect site.

[0030] Secondly, Tremella fuciformis polysaccharide possesses excellent antioxidant activity, reducing oxidative damage to cells by scavenging free radicals and preventing tumor development. Simultaneously, Tremella fuciformis polysaccharide can inhibit tumor cell proliferation and metastasis, and induce tumor cell apoptosis, thus exhibiting good anti-tumor activity. This invention uses Tremella fuciformis polysaccharide gel with high bonding strength (300-500 MPa) and grafts it with antioxidants, forming a polymer through a grafting reaction that covalently bonds between the antioxidant and Tremella fuciformis polysaccharide. This polymer enhances the antioxidant properties of Tremella fuciformis polysaccharide by regulating gelling enzyme activity, thereby delaying the anti-tumor cycle of Tremella fuciformis polysaccharide. Furthermore, the bonding strength is further improved by the hydrogen and ionic bonds formed between the hydroxyl groups in the grafted Tremella fuciformis polysaccharide gel and calcium ions on the surface of porous calcium phosphate through the grafting of antioxidants.

[0031] Thirdly, the porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogel bone binder prepared in this invention has a bonding strength of 400-500 MPa, which is higher than that of traditional bone binders PMMA (40-50 MPa) and nitrate gel (300-375 MPa).

[0032] Fourth, the porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogel bone binder of this invention was obtained through loading antitumor drugs and release experiments. Its antitumor cycle and ability are significantly improved compared with existing composite bone binders (antitumor cycle 1-8 months). In the treatment of bone tumors, this invention is directly applied to the bone tumor resection area, promoting new bone formation and bone repair through integration with human bone, which is an open bone tumor treatment method. Detailed Implementation

[0033] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0034] Example 1

[0035] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0036] Step 1, Preparation of porous calcium phosphate: First, calcium phosphate powder was prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate was reacted with anhydrous calcium chloride at a mass ratio of 1.5:1 to obtain calcium phosphate precipitate, which was then allowed to stand, filtered, and dried in a drying oven at 30℃ for 6 hours to obtain calcium phosphate powder. Next, 40% polyvinyl alcohol was dissolved in deionized water to obtain solution A. 0.5g of calcium phosphate powder was ultrasonically dispersed in anhydrous ethanol for 5 minutes and added to solution A, followed by ultrasonication for another 15 minutes. After completion, the mixture was allowed to stand at room temperature for 12 hours, and the supernatant was removed. The mixture was then centrifuged and washed three times with deionized water, dried at 80℃ for 24 hours, and sieved to obtain porous calcium phosphate powder.

[0037] Step 2, Preparation of 5% Antioxidant-Grafted Tremella Polysaccharide Hydrogel by Free Radical Polymerization: First, add 1g of Tremella polysaccharide to 100mL of deionized water. Simultaneously, add hydrochloric acid solution dropwise to adjust the pH to 3, and heat in a 40℃ water bath for 30min to dissolve, obtaining Tremella polysaccharide gel. Next, add 5% ascorbic acid to graft the Tremella polysaccharide gel, obtaining a mixture; heat the mixture at 40℃ and stir for 30min to obtain 5% antioxidant-grafted Tremella polysaccharide hydrogel.

[0038] Step 3, Preparation of porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant polyvinylpyrrolidone (3%) and crosslinking agent methacrylamide (6%) were dissolved in 80 mL of deionized water by stirring for 5 min, and initiator 2,2'-azobisisobutyronitrile (1%) was dissolved in 40 mL of anhydrous ethanol by stirring for 5 min. Next, 1 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in the polyvinylpyrrolidone solution to obtain the first solution. Subsequently, the first solution, methacrylamide solution, and 2,2'-azobisisobutyronitrile solution were heated to 65 °C and stirred for 4 h to obtain porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. Porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was dried at -50℃ and 30Pa for 36 hours using a freeze-drying method to obtain dried porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0039] Example 2

[0040] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0041] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.5:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 30℃ for 6 hours to obtain calcium phosphate powder. Next, 40% polyacrylic acid is dissolved in deionized water to obtain solution A. 0.5g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 5 minutes and added to solution A, followed by ultrasonication for another 15 minutes. After completion, the mixture is allowed to stand at room temperature for 12 hours, the supernatant is removed, and the mixture is centrifuged and washed three times with deionized water. It is then dried at 80℃ for 24 hours and sieved to obtain porous calcium phosphate powder.

[0042] Step 2: Preparation of 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, add 2g of Tremella fuciformis polysaccharide to 200mL of deionized water. Simultaneously, add hydrochloric acid solution dropwise to adjust the pH to 3, and heat in a 40℃ water bath for 30min to dissolve, obtaining Tremella fuciformis polysaccharide gel. Next, add 10% ascorbic acid to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; heat the mixture at 40℃ and stir for 30min to obtain 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0043] Step 3, Preparation of porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant polyvinylpyrrolidone (3%) and crosslinking agent methacrylamide (6%) were dissolved in 80 mL of deionized water by stirring for 5 min, and initiator 2,2'-azobisisobutyronitrile (1%) was dissolved in 40 mL of anhydrous ethanol by stirring for 5 min. Next, 1 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in the polyvinylpyrrolidone solution to obtain the first solution. Subsequently, the first solution, methacrylamide solution, and 2,2'-azobisisobutyronitrile solution were heated to 65 °C and stirred for 4 h to obtain porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. Porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was dried at -50℃ and 30Pa for 36 hours using a freeze-drying method to obtain dried porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0044] Example 3

[0045] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0046] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.6:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 40℃ for 5.5 h to obtain calcium phosphate powder. Next, 50% polyvinyl alcohol is dissolved in deionized water to obtain solution A. 1.0 g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 10 min and added to solution A, and ultrasonication is continued for another 20 min. After completion, the mixture is allowed to stand at room temperature for 16 h, and the supernatant is removed. The mixture is then centrifuged and washed 4 times with deionized water, dried at 90℃ for 20 h, and sieved to obtain porous calcium phosphate powder.

[0047] Step 2: Preparation of 5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, add 4g of Tremella fuciformis polysaccharide to 400mL of deionized water. Simultaneously, add hydrochloric acid solution dropwise to adjust the pH to 4, and heat in a 45℃ water bath for 25min to dissolve, obtaining Tremella fuciformis polysaccharide gel. Next, add 5% (w / w) tea polyphenols to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; heat and stir the mixture at 45℃ for 25min to obtain 5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0048] Step 3, Preparation of porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant polyacrylic acid (4%) and crosslinking agent dimethacrylamide (6.5%) were dissolved in 90 mL of deionized water for 5 min, and initiator ammonium persulfate (2%) was dissolved in 45 mL of anhydrous ethanol for 5 min. Next, the porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in the polyacrylic acid solution to obtain the first solution. Subsequently, the first solution was heated with the dimethacrylamide solution and the ammonium persulfate solution at 65℃ and stirred for 3.5 h to obtain porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was freeze-dried at -60℃ and 25 Pa for 24 h to obtain dried porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0049] Example 4

[0050] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0051] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.6:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 40℃ for 5.5 h to obtain calcium phosphate powder. Next, 50% polyacrylic acid is dissolved in deionized water to obtain solution A. 1.0 g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 10 min and added to solution A, and ultrasonication is continued for another 20 min. After completion, the mixture is allowed to stand at room temperature for 16 h, the supernatant is removed, and the powder is centrifuged and washed four times with deionized water. It is then dried at 90℃ for 20 h and sieved to obtain porous calcium phosphate powder.

[0052] Step 2: Preparation of 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, add 4g of Tremella fuciformis polysaccharide to 400mL of deionized water. Simultaneously, add hydrochloric acid solution dropwise to adjust the pH to 4, and heat in a 45℃ water bath for 25min to dissolve, obtaining Tremella fuciformis polysaccharide gel. Next, add 10% (w / w) tea polyphenols to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; heat and stir the mixture at 45℃ for 25min to obtain 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0053] Step 3, Preparation of porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant polyacrylic acid (4%) and crosslinking agent dimethacrylamide (6.5%) were dissolved in 90 mL of deionized water for 10 min, and initiator ammonium persulfate (2%) was dissolved in 45 mL of anhydrous ethanol for 10 min. Next, 1.4 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in the polyacrylic acid solution to obtain the first solution. Subsequently, the first solution was heated with the dimethacrylamide solution and the ammonium persulfate solution at 70 °C and stirred for 3.5 h to obtain porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was freeze-dried at -60 °C and 25 Pa for 24 h to obtain dried porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0054] Example 5

[0055] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0056] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.7:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a 50℃ drying oven for 5 hours to obtain calcium phosphate powder. Next, 60% polyvinyl alcohol is dissolved in deionized water to obtain solution A. 1.2g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 15 minutes and added to solution A, and ultrasonication is continued for another 25 minutes. After completion, the mixture is allowed to stand at room temperature for 20 hours, the supernatant is removed, and the powder is centrifuged and washed 5 times with deionized water. It is then dried at 100℃ for 16 hours and sieved to obtain porous calcium phosphate powder.

[0057] Step 2, Preparation of 5% Antioxidant-Grafted Tremella Polysaccharide Hydrogel by Free Radical Polymerization: First, 6g of Tremella polysaccharide was added to 600mL of deionized water. Simultaneously, hydrochloric acid solution was added dropwise to adjust the pH to 5, and the mixture was heated in a 45℃ water bath for 25min to dissolve, obtaining the Tremella polysaccharide gel. Next, 5% (w / w) of flavonoids was added to graft the Tremella polysaccharide gel, obtaining a mixture; the mixture was then heated and stirred at 45℃ for 25min to obtain the 5% antioxidant-grafted Tremella polysaccharide hydrogel.

[0058] Step 3, Preparation of porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant sodium dodecyl sulfate (4.5%) and crosslinking agent methacrylamide (7%) were dissolved in 110 mL of deionized water by stirring for 10 min, and initiator 2,2'-azobisisobutyronitrile (3%) was dissolved in 50 mL of anhydrous ethanol by stirring for 10 min. Next, 1.8 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in sodium dodecyl sulfate solution to obtain the first solution. Subsequently, the first solution was heated with methacrylamide solution and 2,2'-azobisisobutyronitrile solution at 70 °C and stirred for 3 h to obtain porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. Porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was dried at -70℃ and 20 Pa for 20 h using a freeze-drying method to obtain dried porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0059] Example 6

[0060] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0061] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.7:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 50℃ for 5 hours to obtain calcium phosphate powder. Next, 60% polyacrylic acid is dissolved in deionized water to obtain solution A. 1.2g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 15 minutes and added to solution A, followed by ultrasonication for another 25 minutes. After completion, the mixture is allowed to stand at room temperature for 20 hours, the supernatant is removed, and the powder is centrifuged and washed 5 times with deionized water. It is then dried at 100℃ for 16 hours and sieved to obtain porous calcium phosphate powder.

[0062] Step 2: Preparation of 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, 6g of Tremella fuciformis polysaccharide was added to 600mL of deionized water. Simultaneously, hydrochloric acid solution was added dropwise to adjust the pH to 5, and the mixture was heated in a 45℃ water bath for 25min to dissolve, obtaining the Tremella fuciformis polysaccharide gel. Next, 10% (w / w) of flavonoids was added to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; the mixture was then heated and stirred at 45℃ for 25min to obtain the 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0063] Step 3, Preparation of porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant sodium dodecyl sulfate (4.5%) and crosslinking agent methacrylamide (7%) were dissolved in 110 mL of deionized water by stirring for 10 min, and initiator 2,2'-azobisisobutyronitrile (3%) was dissolved in 50 mL of anhydrous ethanol by stirring for 10 min. Next, 1.8 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in sodium dodecyl sulfate solution to obtain the first solution. Subsequently, the first solution, methacrylamide solution, and 2,2'-azobisisobutyronitrile solution were heated to 70 °C and stirred for 3 h to obtain porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. Porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was dried at -70℃ and 20 Pa for 20 h using a freeze-drying method to obtain dried porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0064] Example 7

[0065] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0066] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.8:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 60℃ for 4 hours to obtain calcium phosphate powder. Next, 70% polyacrylic acid is dissolved in deionized water to obtain solution A. 1.5g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 20 minutes and added to solution A, followed by ultrasonication for another 30 minutes. After completion, the mixture is allowed to stand at room temperature for 24 hours, and the supernatant is removed. The mixture is then centrifuged and washed 6 times with deionized water, dried at 120℃ for 12 hours, and sieved to obtain porous calcium phosphate powder.

[0067] Step 2: Preparation of 5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, 8g of Tremella fuciformis polysaccharide was added to 800mL of deionized water. Simultaneously, hydrochloric acid solution was added dropwise to adjust the pH to 6, and the mixture was heated in a 50℃ water bath for 15min to dissolve, obtaining Tremella fuciformis polysaccharide gel. Next, 5% (w / w) tocopherol was added to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; the mixture was heated and stirred at 50℃ for 15min to obtain 5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0068] Step 3, Preparation of porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant sodium dodecylbenzenesulfonate (5%) and crosslinking agent dimethacrylamide (8%) were dissolved in 120 mL of deionized water for 15 min, and initiator ammonium persulfate (4%) was dissolved in 60 mL of anhydrous ethanol for 15 min. Next, 2 g of porous calcium phosphate powder obtained in Step 1 was uniformly dispersed in sodium dodecylbenzenesulfonate solution to obtain the first solution. Subsequently, the first solution was heated with dimethacrylamide solution and ammonium persulfate solution at 75℃ and stirred for 2 h to obtain porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was freeze-dried at -80℃ and 10 Pa for 12 h to obtain dried porous calcium phosphate@5% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0069] Example 8

[0070] A method for preparing a long-acting antitumor organic and inorganic bone binder, the specific process of which is as follows:

[0071] Step 1, Preparation of Porous Calcium Phosphate by Template Method: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate and anhydrous calcium chloride are reacted at a mass ratio of 1.8:1 to obtain calcium phosphate precipitate, which is then allowed to stand, filtered, and dried in a drying oven at 60℃ for 4 hours to obtain calcium phosphate powder. Next, 70% polyacrylic acid is dissolved in deionized water to obtain solution A. 1.5g of calcium phosphate powder is ultrasonically dispersed in anhydrous ethanol for 20 minutes and added to solution A, followed by ultrasonication for another 30 minutes. After completion, the mixture is allowed to stand at room temperature for 24 hours, and the supernatant is removed. The mixture is then centrifuged and washed 6 times with deionized water, dried at 120℃ for 12 hours, and sieved to obtain porous calcium phosphate powder.

[0072] Step 2: Preparation of 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel by free radical polymerization: First, add 8g of Tremella fuciformis polysaccharide to 800mL of deionized water. Simultaneously, add hydrochloric acid solution dropwise to adjust the pH to 6, and heat in a 50℃ water bath for 15min to dissolve, obtaining Tremella fuciformis polysaccharide gel. Next, add 10% tocopherol to graft the Tremella fuciformis polysaccharide gel, obtaining a mixture; heat the mixture at 50℃ and stir for 15min to obtain 10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel.

[0073] Step 3, Preparation of porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel: First, dispersant sodium dodecylbenzenesulfonate (5%) and crosslinking agent dimethacrylamide (8%) were dissolved in 120 mL of deionized water by stirring for 15 min, and initiator ammonium persulfate (4%) was dissolved in 60 mL of anhydrous ethanol by stirring for 15 min. Next, 2 g of porous calcium phosphate powder obtained in Step 2 was uniformly dispersed in the sodium dodecylbenzenesulfonate solution to obtain the first solution. Subsequently, the first solution, dimethacrylamide solution, and ammonium persulfate solution were heated to 75 °C and stirred for 2 h to obtain porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. Porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel was dried at -80℃ and 10 Pa for 12 h using a freeze-drying method to obtain dried porous calcium phosphate@10% antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which was then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

[0074] The bonding strength, antitumor cycle, and antitumor ability of the long-acting antitumor organic and inorganic bone binders in the examples are compared with those of existing bone binders such as nitrate gel and PMMA, as shown in Table 1.

[0075] Table 1 Comparison of the bonding strength and degradation period of the present invention with nitrate gel and PMMA

[0076]

[0077] As shown in the table above, compared with existing bone binders such as nitrate gel and PMMA, the long-acting antitumor organic and inorganic bone binder prepared in this invention uses porous calcium phosphate. Through the regulation of the porous structure and calcium phosphate composition, calcium phosphate degradation is achieved, thereby slowly releasing calcium ions, improving osteogenic properties, and ultimately achieving high bonding strength with human bone at the postoperative bone tumor defect site. Simultaneously, leveraging the high bonding strength and long-acting antitumor properties of Tremella fuciformis polysaccharide hydrogel grafted with one or two of the antioxidants ascorbic acid, tea polyphenols, flavonoids, and tocopherols, porous calcium phosphate is combined with the antioxidant-grafted Tremella fuciformis polysaccharide hydrogel to form a covalently cross-linked network structure, further improving the bonding strength of the bone binder and promoting osteogenic properties with host bone tissue, providing a long-lasting antitumor cycle. This represents a significant improvement compared to both nitrate gel and PMMA. In summary, the preparation method of the organic-inorganic composite bone binder of this invention has high bonding strength, long-lasting anti-tumor cycle and ability, and is expected to have broad application prospects in the field of biomedical materials technology.

[0078] The above description is merely an embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principle of the present invention should be included within the scope of the claims of the present invention.

Claims

1. A method for preparing a long-acting antitumor organic and inorganic bone binder, characterized in that, Includes the following steps: Step 1, Preparation of porous calcium phosphate: First, calcium phosphate powder is prepared by chemical precipitation. Disodium hydrogen phosphate dodecahydrate is reacted with anhydrous calcium chloride to obtain calcium phosphate precipitates with different calcium-to-phosphorus ratios. After standing and filtration, the precipitates are dried to obtain calcium phosphate powder. Polyvinyl alcohol or polyacrylic acid is dissolved in deionized water to obtain solution A as a pore-forming agent. The calcium phosphate powder is dispersed in anhydrous ethanol and solution A is added for ultrasonication. After completion, the powder is allowed to stand and the supernatant is removed. The powder is then washed by centrifugation with deionized water, dried, and sieved to obtain porous calcium phosphate powder. Step 2, Preparation of antioxidant-grafted Tremella polysaccharide hydrogel by free radical polymerization: First, add 1-8g of Tremella polysaccharide to 100-800mL of deionized water to obtain a Tremella polysaccharide solution; simultaneously, add hydrochloric acid solution dropwise, then heat in a water bath at 40-50℃ for 15-30min to dissolve, obtaining a Tremella polysaccharide gel; second, add an antioxidant to graft the Tremella polysaccharide gel to obtain a mixture; stir the mixture to obtain an antioxidant-grafted Tremella polysaccharide gel; the antioxidants selected are one or two of ascorbic acid, tea polyphenols, flavonoids, and tocopherols; Step 3, Preparation of porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogel: First, one of the following dispersants—polyvinylpyrrolidone, polyacrylic acid, sodium dodecyl sulfate, and sodium dodecylbenzene sulfonate—and the crosslinking agent—methacrylamide or dimethacrylamide—are stirred and dissolved in 80-120 mL of deionized water for 5-15 min to obtain dispersant solution and crosslinking agent solution, respectively; the initiator is stirred and dissolved in 40-60 mL of anhydrous ethanol for 5-15 min to obtain initiator solution; second, the hydrogel prepared in step 1... The porous calcium phosphate powder obtained in step 2 is uniformly dispersed in a dispersant solution to obtain a first solution. Subsequently, the first solution is heated and stirred with a crosslinking agent solution, an initiator solution, and the antioxidant-grafted Tremella fuciformis polysaccharide gel obtained in step 2 to obtain a porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel. The porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel is prepared by freeze-drying to obtain a dried porous calcium phosphate@antioxidant-grafted Tremella fuciformis polysaccharide hydrogel powder, which is then sieved to obtain a long-acting anti-tumor organic and inorganic bone binder.

2. The method for preparing a long-acting antitumor organic and inorganic bone binder according to claim 1, characterized in that, In step 1, the mass ratio of disodium hydrogen phosphate dodecahydrate to anhydrous calcium chloride ranges from 1.5:1 to 1.8:1, the drying temperature is 30 to 60°C, and the drying time is 4 to 6 hours. The pore-forming agent contains 40%, 50%, 60%, or 70% polyvinyl alcohol or polyacrylic acid by mass fraction. In step 1, calcium phosphate powder is ultrasonically dispersed uniformly in anhydrous ethanol and added to solution A for further ultrasonication. The mass of calcium phosphate powder is 0.5~1.5g, ultrasonication is performed for 5~20min, and ultrasonication is continued for 15~30min after adding solution A. In step 1, during the standing, centrifugation, washing and drying process, the plant is left to stand at room temperature for 12-24 hours, centrifuged and washed 3-6 times, dried at 80-120℃, and dried for 12-24 hours. The porous calcium phosphate powder obtained in step 1 has a pore size range of 10~100μm and a porosity range of 40%~80%.

3. The method for preparing a long-acting antitumor organic and inorganic bone binder according to claim 1, characterized in that, In step 2, the pH of the Tremella polysaccharide solution is adjusted to 3-6 using hydrochloric acid; The antioxidant has a mass fraction of 5% or 10%.

4. The method for preparing a long-acting antitumor organic and inorganic bone binder according to claim 1, characterized in that, In step 3, the dispersant has a mass fraction range of 3% to 5%; the crosslinking agent has a mass fraction range of 6% to 8%; and the initiator is 2,2'-azobisisobutyronitrile or ammonium persulfate, with a mass fraction range of 1% to 4%. The mass of porous calcium phosphate powder in step 3 is 1~2g; In step 3, the first solution, the crosslinking agent solution, and the initiator solution are heated to a temperature of 65-75°C for 2-4 hours. In step 3, porous calcium phosphate@antioxidant grafted Tremella polysaccharide hydrogels are prepared by freeze-drying. The freezing temperature is -50℃ to -80℃, the vacuum degree is 10 to 30 Pa, and the drying time is 12h to 36h.

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