An injectable hydrogel, its preparation method and application

Abstract

This invention discloses an injectable hydrogel, its preparation method, and its application. The preparation method includes providing solution A and solution B, which can be mixed to obtain the hydrogel. Solution A comprises dispersing polydopamine in a solution of recombinant collagen containing grafted hydrazide groups. Solution B comprises dispersing selenium and magnesium-doped hydrophilic hydroxyapatite in a solution containing chondroitin sulfate. The injectable hydrogel of this invention effectively combines the photothermal effect of polydopamine, the tumor-inhibiting and cell-growth-promoting properties of selenium and magnesium-doped hydroxyapatite, the good biocompatibility of chondroitin sulfate, and the cell-growth-promoting properties of recombinant collagen. Under mild photothermal conditions, it achieves ablation-bone tissue repair integrated treatment of osteosarcoma, exhibiting the characteristics of rapid tumor ablation and bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to an injectable hydrogel, its preparation method, and its application. Background Technology

[0002] Osteosarcoma is a primary malignant bone tumor that commonly occurs in children and adolescents. Osteosarcoma is often accompanied by localized intermittent swelling, pain, and difficulty walking, severely impacting patients' daily lives. Surgical resection is currently the common clinical strategy for treating osteosarcoma. However, incomplete elimination of osteosarcoma cells increases the likelihood of tumor recurrence and metastasis. Furthermore, bone defects caused by osteosarcoma invading normal bone also affect patients' daily lives.

[0003] Photothermal therapy (PTT) is a tumor treatment method that converts light energy into heat energy through photothermal transducers. It is currently widely used and provides a hydrogel that can be combined with photothermal therapy to treat osteosarcoma. It is one of the effective ways to quickly kill tumor cells and reduce damage to healthy tissues. Summary of the Invention

[0004] The technical problem to be solved by this invention is to address the shortcomings of the prior art by providing an injectable hydrogel, its preparation method, and its applications. The injectable hydrogel of this invention effectively combines the photothermal effect of polydopamine, the tumor-inhibiting and cell-growth-promoting properties of selenium and magnesium-doped hydroxyapatite, the good biocompatibility of chondroitin sulfate, and the cell-growth-promoting properties of recombinant collagen. Under mild photothermal conditions, it achieves ablation-bone tissue repair integrated treatment for osteosarcoma, exhibiting the characteristics of rapid tumor ablation and bone defect repair.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a method for preparing an injectable hydrogel, characterized in that it includes: providing liquid A and liquid B, which can be mixed to obtain the hydrogel, wherein liquid A comprises dispersing polydopamine in a solution of recombinant collagen containing grafted hydrazide groups to obtain liquid A; and liquid B comprises dispersing Se and Mg co-doped hydrophilic hydroxyapatite in a solution containing chondroitin sulfate to obtain liquid B.

[0006] The above-mentioned method for preparing an injectable hydrogel is characterized in that, in solution A, the concentration of polydopamine is 0.05-0.15 g / mL, and the concentration of recombinant collagen with grafted hydrazide groups is 0.1-0.8 g / mL; in solution B, the concentration of Se and Mg co-doped hydrophilic hydroxyapatite is 0.05-0.4 g / mL, and the concentration of chondroitin sulfate is 0.1-0.4 g / mL; the volume of solution B is 0.2-1 times the volume of solution A; and the length of the Se and Mg co-doped hydrophilic hydroxyapatite is 150-300 nm, and the width is 5-15 nm.

[0007] The above-mentioned method for preparing an injectable hydrogel is characterized in that the method for preparing the Se and Mg co-doped hydrophilic hydroxyapatite includes:

[0008] An aqueous solution of CaCl2, an aqueous solution of Na2SeO3, and an aqueous solution of soluble phosphate were sequentially dissolved in a methanol solution of oleic acid to obtain a mixture A.

[0009] Under sealed conditions, the mixture A is reacted at 120-180°C for 8-15 hours. An aqueous solution of MgCl2 is added, and the mixture is stirred for 10-30 minutes. Then, it is reacted again at 120-180°C under sealed conditions for 8-15 hours.

[0010] The precipitate in the reaction system was dispersed in cyclohexane, sodium oleate aqueous solution was added, stirred for 10-20 min, and centrifuged to obtain Se and Mg co-doped hydrophilic hydroxyapatite.

[0011] The above-mentioned method for preparing an injectable hydrogel is characterized in that, in the oleic acid methanol solution, the volume of methanol is 3 to 7 times the volume of oleic acid; the molar ratio of Ca / (P+Se) is 1.67, and the molar ratio of Se / (P+Se) is 0.05 to 0.4; the soluble phosphate is Na2HPO4, Na3PO4·12H2O, NaH2PO4, KH2PO4, or K2HPO4; the molar ratio of Mg / Ca is 0.1 to 0.5; the volume of cyclohexane is 10 to 100 times the mass of the precipitate, wherein the volume of cyclohexane is in mL and the mass of the precipitate is in g; and the mass of sodium oleate is 30 to 50 times the mass of the precipitate.

[0012] The above-mentioned method for preparing an injectable hydrogel is characterized in that the method for preparing polydopamine includes: adding dopamine hydrochloride to an aqueous solution of tris(hydroxymethyl)aminomethane, stirring in the dark for 12 to 24 hours, centrifuging, and freeze-drying to obtain polydopamine; wherein the mass of dopamine hydrochloride is 0.15 to 0.5 times the mass of tris(hydroxymethyl)aminomethane.

[0013] The above-mentioned method for preparing an injectable hydrogel is characterized in that the method for preparing the solution of recombinant collagen containing grafted hydrazide groups includes:

[0014] At a pH of 5–6, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were placed in an aqueous solution of recombinant collagen and stirred for 10–30 minutes to activate the carboxyl groups.

[0015] Add adipic acid dihydrazide to the system after activating the carboxyl group, and react for 12–24 hours;

[0016] The reaction mixture was transferred and dialyzed, then freeze-dried to obtain recombinant collagen with grafted hydrazide groups;

[0017] Recombinant collagen with grafted hydrazide groups was dissolved in a buffer solution to obtain a solution of recombinant collagen containing grafted hydrazide groups.

[0018] The above-mentioned method for preparing an injectable hydrogel is characterized in that the mass of 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 to 2 times the mass of recombinant collagen protein, the mass of N-hydroxysuccinimide is 1 to 2 times the mass of recombinant collagen protein, and the mass of adipic acid dihydrazide is 0.67 to 2 times the mass of recombinant collagen protein.

[0019] The above-mentioned method for preparing an injectable hydrogel is characterized in that the preparation method of the solution containing chondroitin sulfate includes:

[0020] Sodium periodate was placed in an aqueous solution of chondroitin sulfate and stirred in the dark for 12–24 hours; the mass of sodium periodate was 1–3 times the mass of chondroitin sulfate.

[0021] Ethylene glycol is added to the reaction system, and the mixture is stirred for 2 to 4 hours to terminate the reaction; the volume of the ethylene glycol is 1.25 to 6.25 times the mass of chondroitin sulfate, and the volume of the ethylene glycol is in mL, while the mass of the chondroitin sulfate is in g.

[0022] After terminating the reaction, the system was dialyzed and lyophilized to obtain chondroitin sulfate oxidized;

[0023] Chondroitin sulfate was dissolved in a buffer solution to obtain a solution containing chondroitin sulfate.

[0024] In addition, the present invention also provides an injectable hydrogel prepared by the above-described method for preparing injectable hydrogels.

[0025] Furthermore, the present invention also provides a method for preparing a gel for osteosarcoma ablation-bone tissue repair and perforation therapy using the above-mentioned injectable hydrogel.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] 1. The injectable hydrogel of the present invention effectively combines the photothermal effect of polydopamine, the tumor-inhibiting and cell-growth-promoting properties of selenium and magnesium-doped hydroxyapatite, the good biocompatibility of chondroitin sulfate, and the cell-growth-promoting properties of recombinant collagen. Based on mild photothermal combination and ion release ablation of tumors, it realizes the treatment of osteosarcoma ablation and bone tissue repair under mild photothermal conditions, and has the characteristics of rapid tumor ablation and bone defect repair.

[0028] 2. The method for preparing the injectable hydrogel of the present invention involves providing solution A and solution B, wherein solution A is a solution containing polydopamine and recombinant collagen containing grafted hydrazide groups, and solution B is a solution containing hydrophilic hydroxyapatite co-doped with Se and Mg and chondroitin sulfate. After mixing solutions A and B, the mixture can be implanted into the osteosarcoma site in a non-invasive or minimally invasive manner. Under the mild photothermal conditions of NIR irradiation (41-45°C), the tumor is killed, thereby achieving ablation of osteosarcoma and repair of bone tissue, while avoiding damage to surrounding normal tissues.

[0029] 3. The method for preparing the injectable hydrogel of the present invention includes using CaCl2, Na2SeO3 and soluble phosphate as main raw materials, adding MgCl2 after a closed hydrothermal reaction, and then performing a closed hydrothermal reaction again to obtain Se and Mg co-doped hydrophilic hydroxyapatite. The obtained hydrogel has the characteristic of effectively promoting the continuous release of metal ions with the effect of killing tumor cells. At the same time, the metal ions can continue to be released after tumor ablation with the degradation of the hydrogel network, thereby accelerating the repair process of bone defects.

[0030] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0031] Figure 1 This is a TEM image of the Se and Mg co-doped hydrophilic hydroxyapatite from Example 1-1.

[0032] Figure 2 XRD of Se and Mg co-doped hydrophilic hydroxyapatite in Example 1-1.

[0033] Figure 3 SEM image of polydopamine from Example 2-1.

[0034] Figure 4 FT-IR of recombinant collagen CF-1552, adipic acid dihydrazide (ADH), and recombinant collagen CF-1552-ADH with grafted hydrazide groups as described in Example 3-1.

[0035] Figure 5 The 1H-NMR spectra of chondroitin sulfate before and after modification in Example 4-1 are shown.

[0036] Figure 6 This is a schematic diagram illustrating the injectability properties of the injectable hydrogel described in Example 5-1.

[0037] Figure 7 This is a schematic diagram of the photothermal properties of the injectable hydrogel described in Example 5-1.

[0038] Figure 8 This is a schematic diagram of the experimental results of the efficacy of the injectable hydrogel on hBMSCs in Example 5-1.

[0039] Figure 9 This is a schematic diagram of the efficacy experiment results of the injectable hydrogel on MG-63 cells in Example 5-1.

[0040] Figure 10 This is an image of AO / EB live and dead cell staining after NIR irradiation of the injectable hydrogel in Example 5-1. Detailed Implementation

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; and the materials and reagents used are commercially available unless otherwise specified.

[0042] Example 1-1

[0043] This embodiment provides a Se and Mg co-doped hydrophilic hydroxyapatite, the preparation method of which includes:

[0044] Step 101: Under magnetic stirring, mix 1 mL of oleic acid and 3 mL of methanol evenly to obtain the precursor solution;

[0045] Step 102: Add 6 mL of 0.25 M CaCl2 aqueous solution and 0.3 mL of 0.15 M Na2SeO3 aqueous solution to the precursor solution, stir for 10 minutes, add 5.7 mL of 0.15 M Na2HPO4 aqueous solution, stir for 10 minutes to obtain mixture A;

[0046] Step 103: Place the mixture A in a polytetrafluoroethylene reactor, seal it, and react it at 120°C for 8 hours. Add 0.6 mL of 0.25 M MgCl2 aqueous solution, stir for 10 minutes, and then react it again at 120°C under sealed conditions for 8 hours.

[0047] Step 104: Cool the reaction system to room temperature and collect the precipitate;

[0048] Step 105: Disperse 0.05g of precipitate in 5mL of cyclohexane, add 15mL of sodium oleate aqueous solution with a concentration of 0.1g / mL, stir for 10 minutes, centrifuge with anhydrous ethanol to aid precipitation, and obtain Se and Mg co-doped hydrophilic hydroxyapatite.

[0049] Examples 1-2

[0050] This embodiment provides a Se and Mg co-doped hydrophilic hydroxyapatite, the preparation method of which includes:

[0051] Step 101: Under magnetic stirring, mix 1 mL of oleic acid and 7 mL of methanol evenly to obtain the precursor solution;

[0052] Step 102: Add 6 mL of 0.25 M CaCl2 aqueous solution and 2.4 mL of 0.15 M Na2SeO3 aqueous solution to the precursor solution, stir for 30 minutes, add 3.6 mL of 0.15 M NaH2PO4 aqueous solution, stir for 30 minutes to obtain mixture A;

[0053] Step 103: Place the mixture A in a polytetrafluoroethylene reactor, seal it, and react it at 180°C for 15 hours. Add 3 mL of 0.25 M MgCl2 aqueous solution, stir for 30 minutes, and then react it again at 180°C under sealed conditions for 15 hours.

[0054] Step 104: Cool the reaction system to room temperature and collect the precipitate;

[0055] Step 105: Disperse 0.5g of precipitate in 5mL of cyclohexane, add 25mL of sodium oleate aqueous solution with a concentration of 1g / mL, stir for 20 minutes, and centrifuge with anhydrous ethanol to aid precipitation to obtain Se and Mg co-doped hydrophilic hydroxyapatite.

[0056] The properties of the Se and Mg co-doped hydrophilic hydroxyapatite in this embodiment are basically the same as those in Example 1-1.

[0057] Examples 1-3

[0058] This embodiment provides a Se and Mg co-doped hydrophilic hydroxyapatite, the preparation method of which includes:

[0059] Step 101: Under magnetic stirring, mix 1 mL of oleic acid and 5 mL of methanol evenly to obtain the precursor solution;

[0060] Step 102: Add 6 mL of 0.25 M CaCl2 aqueous solution and 2.4 mL of 0.15 M Na2SeO3 aqueous solution to the precursor solution, stir for 20 minutes, then add 3.6 mL of 0.15 M soluble phosphate aqueous solution, stir for 20 minutes to obtain mixture A; in this embodiment, the soluble phosphate is Na3PO4·12H2O, which is feasible, and the soluble phosphate can also be KH2PO4 or K2HPO4;

[0061] Step 103: Place the mixture A in a polytetrafluoroethylene reactor, seal it, and react it at 160°C for 10 hours. Add 3 mL of 0.25 M MgCl2 aqueous solution, stir for 20 minutes, and then react it again at 160°C under sealed conditions for 10 hours.

[0062] Step 104: Cool the reaction system to room temperature and collect the precipitate;

[0063] Step 105: Disperse 0.5g of precipitate in 10mL of cyclohexane, add 20mL of sodium oleate aqueous solution with a concentration of 1g / mL, stir for 15 minutes, centrifuge with anhydrous ethanol to aid precipitation, and obtain Se and Mg co-doped hydrophilic hydroxyapatite.

[0064] The properties of the Se and Mg co-doped hydrophilic hydroxyapatite in this embodiment are basically the same as those in Example 1-1.

[0065] Example 2-1

[0066] This embodiment provides a polydopamine, the preparation method of which includes:

[0067] Dissolve 0.15g of tris(hydroxymethyl)aminomethane in 150mL of deionized water, add 75mg of dopamine hydrochloride, stir for 12 hours in the dark, centrifuge, collect the precipitate, freeze-dry the precipitate to obtain polydopamine.

[0068] Example 2-2

[0069] This embodiment provides a polydopamine, the preparation method of which includes:

[0070] Dissolve 2.5g of tris(hydroxymethyl)aminomethane in 250mL of deionized water, add 375mg of dopamine hydrochloride, stir in the dark for 24 hours, centrifuge, collect the precipitate, freeze-dry the precipitate to obtain polydopamine.

[0071] The polydopamine properties in this embodiment are basically the same as those in Example 2-1.

[0072] Example 2-3

[0073] This embodiment provides a polydopamine, the preparation method of which includes:

[0074] Dissolve 1g of tris(hydroxymethyl)aminomethane in 200mL of deionized water, add 300mg of dopamine hydrochloride, stir in the dark for 20 hours, centrifuge, collect the precipitate, freeze-dry the precipitate to obtain polydopamine.

[0075] The polydopamine properties in this embodiment are basically the same as those in Example 2-1.

[0076] Figure 1 TEM images of the Se and Mg co-doped hydrophilic hydroxyapatite from Example 1-1. According to... Figure 1 As can be seen, the Se and Mg co-doped hydrophilic hydroxyapatite is 150-300 nm long and 5-15 nm wide, and is in the shape of nanorods with uniform size.

[0077] Figure 2 XRD pattern of Se and Mg co-doped hydrophilic hydroxyapatite from Example 1-1. Figure 2 As can be seen, the comparison with the hydroxyapatite standard card JCPDS no. 74-0565 shows that the product is hydroxyapatite.

[0078] Figure 3 SEM image of polydopamine from Example 2-1. Figure 3 It can be seen that polydopamine consists of nanospheres with a diameter of 170–300 nm and uniform size.

[0079] Example 3-1

[0080] This embodiment provides a solution of recombinant collagen CF-1552-ADH containing a grafted hydrazide group, the preparation method of which includes:

[0081] Step 301: Under stirring conditions, dissolve 1g of recombinant collagen CF-1552 in 100mL of deionized water, adjust the pH to 5-6 with 1M hydrochloric acid solution, add 1g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2g of N-hydroxysuccinimide, and stir for 10 minutes to activate the carboxyl groups; the recombinant collagen is CF-1552 type recombinant collagen, purchased from Xi'an Giant Biogene Technology Co., Ltd.

[0082] Step 302: Add 0.67g of adipic acid dihydrazide to the system after activating the carboxyl group, and stir the reaction at room temperature for 12 hours;

[0083] Step 303: Transfer the reaction system to a dialysis bag with a cutoff of 3500 Da, dialyze with deionized water for 3 days, changing the deionized water daily during the dialysis process, and freeze-dry the dialysis system to obtain recombinant collagen with grafted hydrazide group (CF-1552-ADH).

[0084] Step 304: Dissolve 0.1g of CF-1552-ADH in 1mL of PBS to obtain a solution of recombinant collagen CF-1552-ADH containing grafted hydrazide groups, which is solution A0.

[0085] Example 3-2

[0086] This embodiment provides a solution of recombinant collagen CF-1552-ADH containing a grafted hydrazide group, the preparation method of which includes:

[0087] Step 301: Under stirring conditions, dissolve 10g of recombinant collagen CF-1552 in 100mL of deionized water, adjust the pH of the solution to 5-6 with 1M hydrochloric acid solution, add 20g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10g of N-hydroxysuccinimide, and stir for 30 minutes to activate the carboxyl groups.

[0088] Step 302: Add 20g of adipate dihydrazide to the system after activating the carboxyl group, and stir at room temperature for 24 hours;

[0089] Step 303: Transfer the reaction system to a dialysis bag with a capacity cutoff of 3500 Da, and dialyze with deionized water for 5 days, changing the deionized water daily during the dialysis process. Freeze-dry the dialysis system to obtain recombinant collagen with grafted hydrazide group (CF-1552-ADH). The performance of the recombinant collagen with grafted hydrazide group in this embodiment is basically the same as that in Example 3-1.

[0090] Step 304: Dissolve 0.8g of CF-1552-ADH in 1mL of PBS to obtain a solution of recombinant collagen CF-1552-ADH containing grafted hydrazide groups.

[0091] Example 3-3

[0092] This embodiment provides a solution of recombinant collagen CF-1552-ADH containing a grafted hydrazide group, the preparation method of which includes:

[0093] Step 301: Under stirring conditions, dissolve 10g of recombinant collagen CF-1552 in 500mL of deionized water, adjust the pH of the solution to 5-6 with 1M hydrochloric acid solution, add 15g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 15g of N-hydroxysuccinimide, and stir for 20 minutes to activate the carboxyl groups.

[0094] Step 302: Add 15g of adipate dihydrazide to the system after activating the carboxyl group, and stir the reaction at room temperature for 20 hours;

[0095] Step 303: Transfer the reaction system to a dialysis bag with a capacity cutoff of 3500 Da, and dialyze with deionized water for 4 days, changing the deionized water daily during the dialysis process. Freeze-dry the dialysis system to obtain recombinant collagen with grafted hydrazide group (CF-1552-ADH). The performance of the recombinant collagen with grafted hydrazide group in this embodiment is basically the same as that in Example 3-1.

[0096] Step 304: Dissolve 0.6g of CF-1552-ADH in 1mL of PBS to obtain a solution of recombinant collagen CF-1552-ADH containing grafted hydrazide groups.

[0097] Example 4-1

[0098] This embodiment provides a solution containing chondroitin sulfate OCS, wherein the chondroitin sulfate is obtained by aldehyde modification of chondroitin sulfate, and its preparation method includes:

[0099] Step 401: Under stirring conditions, dissolve 0.8g of chondroitin sulfate in 80mL of deionized water, add 0.8g of sodium periodate, and stir the mixture in the dark for 12 hours.

[0100] Step 402: Add 5 mL of ethylene glycol to the reaction mixture and stir for 2 hours to terminate the reaction;

[0101] Step 403: After the reaction is terminated, the system is transferred to a dialysis bag with a cutoff of 3500 Da and dialyzed with deionized water for 3 days. The deionized water is changed every day during the dialysis process. The system after dialysis is freeze-dried to obtain chondroitin sulfate (OCS).

[0102] Step 404: Dissolve 0.1g of OCS in 1mL of PBS to obtain a solution containing chondroitin sulfate OCS, denoted as solution B0.

[0103] Figure 4 FT-IR of recombinant collagen CF-1552, adipic acid dihydrazide (ADH), and grafted hydrazide group of recombinant collagen CF-1552-ADH from Example 3-1. Figure 4It can be seen that 2924cm -1 and 2862cm -1 The presence of a characteristic absorption peak belonging to the amino group indicates that the grafting of the hydrazide group was successful.

[0104] Example 4-1 1H-NMR of chondroitin sulfate before and after modification: Figure 5 As shown, according to Figure 5 As can be seen, compared with CS, new characteristic peaks appear at 8.1 and 8.3 ppm in the OCS curve, indicating that the modification of OCS was successful.

[0105] Example 4-2

[0106] This embodiment provides a solution containing chondroitin sulfate (OCS), the preparation method of which includes:

[0107] Step 401: Under stirring conditions, dissolve 8g of chondroitin sulfate in 80mL of deionized water, add 24g of sodium periodate, and stir the mixture in the dark for 24 hours.

[0108] Step 402: Add 10 mL of ethylene glycol to the reaction mixture and stir for 4 hours to terminate the reaction;

[0109] Step 403: Transfer the system after the reaction is terminated to a dialysis bag with a capacity of 3500 Da, and dialyze with deionized water for 5 days. Change the deionized water every day during the dialysis process. Freeze-dry the system after dialysis to obtain oxychloride chondroitin sulfate (OCS). The performance of oxychloride chondroitin sulfate in this embodiment is basically the same as that in Example 4-1.

[0110] Step 404: Dissolve 0.4g of OCS in 1mL of PBS to obtain a solution containing chondroitin sulfate OCS.

[0111] Example 4-3

[0112] This embodiment provides a solution containing chondroitin sulfate (OCS), the preparation method of which includes:

[0113] Step 401: Under stirring conditions, dissolve 2g of chondroitin sulfate in 80mL of deionized water, add 4g of sodium periodate, and stir the reaction in the dark for 20 hours.

[0114] Step 402: Add 8 mL of ethylene glycol to the reaction mixture and stir for 3 hours to terminate the reaction;

[0115] Step 403: Transfer the system after the reaction is terminated to a dialysis bag with a capacity of 3500 Da, and dialyze with deionized water for 4 days. Change the deionized water every day during the dialysis process. Freeze-dry the system after dialysis to obtain oxychloride chondroitin sulfate (OCS). The performance of oxychloride chondroitin sulfate in this embodiment is basically the same as that in Example 4-1.

[0116] Step 404: Dissolve 0.2g of OCS in 1mL of PBS to obtain a solution containing chondroitin sulfate OCS.

[0117] Example 5-1

[0118] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0119] Step 501: Disperse 0.05g of the Se and Mg co-doped hydrophilic hydroxyapatite from Example 1-1 in 1mL of Solution B0 from Example 4-1 to obtain Solution B;

[0120] Step 502: Disperse 0.05g of polydopamine from Example 2-1 in 1mL of solution A0 from Example 3-1 to obtain solution A;

[0121] Step 503: Quickly mix 1 mL of solution A and 1 mL of solution B to obtain an injectable hydrogel; in practical applications, solution A and solution B can be injected into the osteosarcoma site to form a hydrogel in situ.

[0122] Example 5-2

[0123] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0124] Step 501: Disperse 0.4g of the Se and Mg co-doped hydrophilic hydroxyapatite from Example 1-1 in 1mL of Solution B0 from Example 4-1 to obtain Solution B;

[0125] Step 502: Disperse 0.15g of polydopamine from Example 2-1 in 1mL of solution A0 from Example 3-1 to obtain solution A;

[0126] Step 503: Quickly mix 1 mL of solution A and 0.2 mL of solution B to obtain an injectable hydrogel; in practical applications, solution A and solution B can be injected into the osteosarcoma site to form a hydrogel in situ.

[0127] The properties of the injectable hydrogel in this embodiment are basically the same as those in Example 5-1.

[0128] Example 5-3

[0129] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0130] Step 501: Disperse 0.2g of Se and Mg co-doped hydrophilic hydroxyapatite from Examples 1-2 in 1mL of Solution B0 from Examples 4-2 to obtain Solution B;

[0131] Step 502: Disperse 0.1g of polydopamine from Example 2-2 in 1mL of solution A0 from Example 3-2 to obtain solution A;

[0132] Step 503: Quickly mix 1 mL of solution A and 0.5 mL of solution B to obtain an injectable hydrogel; in practical applications, solution A and solution B can be injected into the osteosarcoma site to form a hydrogel in situ.

[0133] The properties of the injectable hydrogel in this embodiment are basically the same as those in Example 5-1.

[0134] Example 5-4

[0135] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0136] Step 501: Disperse 0.2g of Se and Mg co-doped hydrophilic hydroxyapatite from Examples 1-3 in 1mL of Solution B0 from Examples 4-3 to obtain Solution B;

[0137] Step 502: Disperse 0.12g of polydopamine from Examples 2-3 in 1mL of solution A0 from Examples 3-3 to obtain solution A;

[0138] Step 503: Quickly mix 1 mL of solution A and 0.6 mL of solution B to obtain an injectable hydrogel; in practical applications, solution A and solution B can be injected into the osteosarcoma site to form a hydrogel in situ.

[0139] Performance Evaluation

[0140] Figure 6 This is a schematic diagram illustrating the injectability of the injectable hydrogel described in Example 5-1. The injectable hydrogel was loaded into a syringe and injected into the target site. The results show that the hydrogel of the present invention can be injected and filled, and can gel in situ at the target site.

[0141] Figure 7 This is a schematic diagram of the photothermal properties of the injectable hydrogel described in Example 5-1. The testing method includes using NIR (808nm, 1.5W / cm²). 2 The hydrogel was irradiated for different durations, and the temperature of the hydrogel was tested. The results showed that the temperature of the injectable hydrogel of the present invention increased with the extension of the irradiation time, indicating that the hydrogel of the present invention has excellent heating effect.

[0142] Cellular Experiment

[0143] Using the injectable hydrogel described in Example 5-1 as the research object, its efficacy in promoting the growth of normal cells (mesenchymal stem cells, hBMSCs) and inhibiting or killing cancer cells (osteosarcoma cells, MG-63) was investigated, specifically including:

[0144] (1) Preparation of DMEM extract for injectable hydrogels

[0145] The injectable hydrogel was freeze-dried, sterilized by Co-60 irradiation, and then soaked in DMEM complete culture medium at a concentration of 0.1 g / mL for 3 days (37°C) to obtain the hydrogel extract. Before use, the extract was filtered through a filter membrane to obtain the DMEM extract of the injectable hydrogel.

[0146] (2) Determination of the cytotoxicity of injectable hydrogels against hBMSCs and MG-63:

[0147] ① Cell plating: using 1×10 4 The cell density was determined by placing the cell dispersion in well plates. In 96-well plates, each well contained 100 μL of cell dispersion for hBMSCs and 1 mL of cell dispersion for MG-63. In 24-well plates, each well contained 1 mL of cell dispersion for hBMSCs and MG-63. The cells were incubated in an incubator (37°C, 5% CO2) for 24 hours.

[0148] ② Cytotoxicity assay: The 96-well plates containing the adhered hBMSCs and MG-63 cells were removed, and the culture medium was replaced with DMEM extract containing injectable hydrogel from step 1. The cells were then cultured in an incubator (37℃, 5% CO2). At 24 and 48 hours of culture, 50 μL of MTT was added to each well, and incubation continued for 2–4 hours. Afterward, all liquid was removed from the wells, and 150 μL of DMSO was added to each well. The absorbance (OD) value of each well was measured at 490 nm using a microplate reader, and cell viability was calculated based on the OD value. The control group consisted of cells cultured in complete DMEM medium.

[0149] ③ Staining of AO / EB live and dead cells: Remove the 24-well plates from ① where MG-63 cells have adhered. Cover the upper part of the cells with 0.5–1 mm of injectable hydrogel, then stain with NIR (808 nm, 1.5 W / cm²). 2 Irradiate the hydrogel for 5 minutes, remove the hydrogel, stain the cells with AO / EB staining solution, and after 20 minutes of staining, observe and photograph the cells under an inverted microscope.

[0150] The experimental results of the efficacy of hydrogel on hBMSCs are as follows: Figure 8 As shown. According to Figure 8 It is evident that the survival rate of hBMSCs was higher than 100% after 24 and 48 hours of culture, indicating that the hydrogel has no cytotoxicity to hBMSCs and can promote the proliferation of hBMSCs and accelerate the repair of bone defects.

[0151] The efficacy of hydrogel on MG-63 cells was experimentally demonstrated as follows: Figure 9 As shown. According to Figure 9 It is evident that the survival rate of MG-63 cells was less than 100% after 24 and 48 hours of cell culture. This may be because the release of Se from the Se and Mg co-doped hydrophilic hydroxyapatite inhibited the proliferation of MG-63 cells.

[0152] The experimental results of the photothermal effect of hydrogel on MG-63 cells are as follows: Figure 10 As shown. According to Figure 10 As can be seen in the figure, the red fluorescence of the cells indicates that the injectable hydrogel of the present invention has a photothermal effect that can kill a large number of MG-63 cells and has a tumor ablation effect.

[0153] comprehensive Figures 8-10 The results of the cytological experiments show that the injectable hydrogel of the present invention can effectively achieve postoperative repair treatment of osteosarcoma based on the treatment strategy of osteosarcoma ablation-bone tissue repair. It can not only inhibit the growth of osteosarcoma cells and ablate the tumor, but also promote the growth of hBMSCs and promote the formation of defective bone.

[0154] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the present invention. Any simple modifications, alterations, or equivalent structural changes made to the above embodiments based on the technical essence of the invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing an injectable hydrogel, characterized by, The application relates to a method for preparing a hydrogel, and a hydrogel prepared by the method. The method comprises the following steps: providing an A solution and a B solution which can be mixed to obtain the hydrogel, wherein the A solution is prepared by dispersing polydopamine in a solution containing recombinant collagen with grafted hydrazide groups; and the B solution is prepared by dispersing Se and Mg co-doped hydrophilic hydroxyapatite in a solution containing oxidized chondroitin sulfate; The method for preparing the Se and Mg co-doped hydrophilic hydroxyapatite comprises the following steps: An aqueous solution of CaCl2, an aqueous solution of Na2SeO3 and an aqueous solution of soluble phosphate are sequentially dissolved in an oleic acid methanol solution to obtain a mixed solution A; Under a sealed condition, the mixed solution A is reacted at a temperature of 120-180 DEG C for 8-15 hours, an aqueous solution of MgCl2 is added, and then stirred for 10-30 minutes; and the reaction is carried out again under a sealed condition at a temperature of 120-180 DEG C for 8-15 hours; The precipitate in the reaction system is dispersed in cyclohexane, an aqueous solution of sodium oleate is added, and then stirred for 10-20 minutes; and centrifugation is carried out to obtain Se and Mg co-doped hydrophilic hydroxyapatite.

2. The method of claim 1, wherein the injectable hydrogel is prepared by mixing the first and second components in a ratio of 1 : 1 to 1 :

3. In the A solution, the concentration of polydopamine is 0.05-0.15 g / mL, and the concentration of the recombinant collagen with grafted hydrazide groups is 0.1-0.8 g / mL; in the B solution, the concentration of the Se and Mg co-doped hydrophilic hydroxyapatite is 0.05-0.4 g / mL, and the concentration of the oxidized chondroitin sulfate is 0.1-0.4 g / mL; the volume of the B solution is 0.2-1 times the volume of the A solution; the length of the Se and Mg co-doped hydrophilic hydroxyapatite is 150-300 nm, and the width is 5-15 nm.

3. The method for preparing an injectable hydrogel according to claim 1, characterized in that, In the oleic acid methanol solution, the volume of methanol is 3-7 times the volume of oleic acid; the molar ratio of Ca / (P+Se) is 1.67, and the molar ratio of Se / (P+Se) is 0.05-0.4; the soluble phosphate is Na2HPO4, Na3PO4.12H2O, NaH2PO4, KH2PO4 or K2HPO4; the molar ratio of Mg / Ca is 0.1-0.5; the volume of cyclohexane is 10-100 times the mass of the precipitate; the unit of the volume of cyclohexane is mL, and the unit of the mass of the precipitate is g; the mass of sodium oleate is 30-50 times the mass of the precipitate.

4. The method of claim 1, wherein the injectable hydrogel is prepared by mixing the first and second components in a ratio of 1:1 to 1:3.

5. The method for preparing the polydopamine comprises the following steps: dopamine hydrochloride is added into a water solution of tris-hydroxymethyl aminomethane, and then stirred in the dark for 12-24 hours; centrifugation is carried out; and freeze-drying is carried out to obtain polydopamine; the mass of the dopamine hydrochloride is 0.15-0.5 times the mass of the tris-hydroxymethyl aminomethane.

5. The method of claim 1, wherein the injectable hydrogel is prepared by mixing the first and second components in a ratio of 1:1 to 1:

3. The method for preparing the solution containing the recombinant collagen with grafted hydrazide groups comprises the following steps: Under the condition of pH 5-6, 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide are placed in a water solution of recombinant collagen, and then stirred for 10-30 minutes to activate carboxyl groups; Hexanedioic acid dihydrazide is added into the system after the activation of the carboxyl groups, and then reacted for 12-24 hours; The system after the reaction is transferred for dialysis, and then freeze-dried to obtain the recombinant collagen with grafted hydrazide groups; Dissolve the recombinant collagen with grafted hydrazide groups in a buffer solution to obtain a solution of the recombinant collagen with grafted hydrazide groups.

6. The method of claim 5, wherein the injectable hydrogel is prepared by mixing the first and second solutions. The 1-ethyl-(3-dimethylaminopropyl)carbodiimide has a mass of 1-2 times the mass of the recombinant collagen, the N-hydroxysuccinimide has a mass of 1-2 times the mass of the recombinant collagen, and the adipic acid dihydrazide has a mass of 0.67-2 times the mass of the recombinant collagen.

7. The method for preparing an injectable hydrogel according to claim 1, characterized in that, The method for preparing the solution containing oxidized chondroitin sulfate comprises: Put sodium periodate in an aqueous solution of chondroitin sulfate, stir and react in the dark for 12-24 hours; the mass of the sodium periodate is 1-3 times the mass of the chondroitin sulfate; Add ethylene glycol to the system after the reaction to terminate the reaction, and stir for 2-4 hours; the volume of the ethylene glycol is 1.25-6.25 times the mass of the chondroitin sulfate, the volume of the ethylene glycol is in mL, and the mass of the chondroitin sulfate is in g; Dialyze the system after the reaction is terminated, and freeze-dry to obtain oxidized chondroitin sulfate; Dissolve the oxidized chondroitin sulfate in a buffer solution to obtain a solution containing oxidized chondroitin sulfate.

8. An injectable hydrogel prepared by the method according to claim 1.

9. A method for preparing a gel for ablation-bone tissue repair through treatment of osteosarcoma by using the injectable hydrogel according to claim 8.

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