Preparation method and application of injectable nanocomposite hydrogel for alleviating osteoarthritis

Abstract

The invention discloses a preparation method of an injectable nanocomposite hydrogel for alleviating osteoarthritis, first synthesizing the hydrophilic hydroxyapatite doped with strontium and europium ions;Secondly, using chondroitin sulfate and adipic acid dihydrazide as main raw materials to prepare the chondroitin sulfate solution of grafted hydrazide groups;Again, using hyaluronic acid, 3-aminophenylboronic acid, sodium periodate as main raw materials to prepare the oxidized hyaluronic acid solution of grafted phenylboronic acid groups;Finally, the hydrophilic hydroxyapatite doped with strontium and europium ions, the chondroitin sulfate solution of grafted hydrazide groups, the oxidized hyaluronic acid solution of grafted phenylboronic acid groups and recombinant collagen solution are blended and stirred to obtain an injectable nanocomposite hydrogel. The injectable nanocomposite hydrogel obtained by the present invention has good biocompatibility and can create a favorable local immune microenvironment to immunomodulate macrophages to alleviate osteoarthritis and promote subchondral bone repair.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an injectable nanocomposite hydrogel for alleviating osteoarthritis, and belongs to the technical field of biomaterials. Background Art

[0002] Articular cartilage is a supporting connective tissue characterized by toughness, viscoelasticity, and lubricity. It plays a crucial role in load-bearing and lubricating joints during movement. Osteoarthritis, characterized by cartilage degeneration and subchondral bone remodeling, affects over 7% of the global population. The knee is the most commonly affected joint, with 365 million people affected, followed by the hand and hip joints. With the accelerated aging population and rising obesity rates in my country, the number of osteoarthritis patients will continue to increase. The pathological characteristics of osteoarthritis are primarily high levels of reactive oxygen species (ROS), an imbalance in the M1 / M2 phenotype of macrophages, bone defects, and impaired synovial fluid. Current treatments still cannot fully meet the complex needs of osteoarthritis treatment. Furthermore, material activity and toxicity, as well as the ability to minimize the inflammatory response of implants and accommodate the irregular morphology of inflammatory sites, remain crucial challenges for the treatment of osteoarthritis. Therefore, the development of an injectable nanocomposite hydrogel with excellent biocompatibility, the ability to reduce ROS levels, reprogram macrophage polarization, promote osteogenesis, and maintain lubrication is crucial for alleviating osteoarthritis. Summary of the Invention

[0003] The present invention addresses the shortcomings of existing technologies by providing a method for preparing an injectable nanocomposite hydrogel for alleviating osteoarthritis. The injectable nanocomposite hydrogel obtained by the present invention has excellent biocompatibility and can create a favorable local immune microenvironment to immunomodulate macrophages, thereby alleviating osteoarthritis and promoting subchondral bone repair.

[0004] The implementation process of the present invention is as follows:

[0005] A method for preparing an injectable nanocomposite hydrogel for relieving osteoarthritis comprises the following steps:

[0006] (1) preparing hydrophilic hydroxyapatite doped with strontium and europium ions, specifically comprising: dissolving aqueous solutions of Ca(NO3)2, Sr(NO3)2, and Eu(NO3)3 and aqueous solutions of soluble phosphates in a methanol solution of methyl oleate in sequence to obtain a mixed solution, reacting the mixed solution in a closed reactor at 100-200°C for 5-15 hours, and collecting the precipitate; dispersing the precipitate in Tris-HCl buffer, adding tannic acid, stirring thoroughly, and collecting the precipitate to obtain hydrophilic hydroxyapatite doped with strontium and europium ions with a length of 150-300 nm and a diameter of 10-15 nm;

[0007] (2) Preparing a chondroitin sulfate solution grafted with a hydrazide group, specifically comprising: dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in an aqueous solution of chondroitin sulfate at a pH of 5 to 6 and stirring the solution to activate the carboxyl group; adding adipic acid dihydrazide to the system after the carboxyl group activation to carry out a full reaction; transferring the system after the reaction to dialyze, freeze-drying, and obtaining a chondroitin sulfate grafted with a hydrazide group; dissolving the chondroitin sulfate grafted with a hydrazide group in a PBS buffer solution to obtain a chondroitin sulfate solution containing a grafted hydrazide group;

[0008] (3) Preparing a hyaluronic acid solution grafted with phenylboronic acid groups, specifically comprising: placing 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in an aqueous solution of hyaluronic acid at a pH of 5 to 6 and stirring thoroughly to activate the carboxyl groups; adding 3-aminophenylboronic acid to the system after the carboxyl groups are activated and reacting thoroughly; transferring the system after the reaction to dialyze, and freeze-drying to obtain hyaluronic acid grafted with phenylboronic acid groups; dissolving the oxidized hyaluronic acid grafted with phenylboronic acid groups in deionized water to obtain an aqueous solution of hyaluronic acid containing grafted phenylboronic acid groups;

[0009] (4) preparing a solution of oxidized hyaluronic acid grafted with phenylboronic acid groups, specifically comprising: dissolving sodium periodate in an aqueous solution of hyaluronic acid grafted with phenylboronic acid groups, adding ethylene glycol after sufficient reaction in the dark, and stirring to terminate the reaction; dialyzing the system after the termination of the reaction, and freeze-drying to obtain oxidized hyaluronic acid grafted with phenylboronic acid groups; dissolving the oxidized hyaluronic acid grafted with phenylboronic acid groups in a PBS buffer solution to obtain an oxidized hyaluronic acid solution containing grafted phenylboronic acid groups;

[0010] (5) Preparing an injectable nanocomposite hydrogel, specifically comprising: uniformly blending the hydrophilic hydroxyapatite doped with strontium and europium ions in step (1), the chondroitin sulfate solution grafted with hydrazide groups in step (2), the oxidized hyaluronic acid solution grafted with phenylboronic acid groups in step (4), and the recombinant collagen solution to obtain the injectable nanocomposite hydrogel.

[0011] The preparation method of the injectable nanocomposite hydrogel for relieving osteoarthritis is as follows: in step (1), in the methanol solution of methyl oleate, the volume of methanol is 3 to 7 times the volume of methyl oleate; the molar ratio of (Ca+Sr+Eu) / P is 1.67, and the molar ratio of Sr / (Ca+Sr+Eu) is 0.05 to 0.5; the soluble phosphate is selected from Na2HPO4, Na3PO4·12H2O, NaH2PO4, KH2PO4 or K2HPO4; the molar ratio of Eu / (Ca+Sr+Eu) is 0.05 to 0.5; the volume of the cyclohexane is 10 to 100 times the mass of the precipitate, the unit of the cyclohexane volume is mL, and the unit of the precipitate mass is g; the mass of the tannic acid is 3 to 6 times the mass of the precipitate.

[0012] In the above-mentioned method for preparing the injectable nanocomposite hydrogel for relieving osteoarthritis, in step (2), the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 to 2 times the mass of chondroitin sulfate, the mass of the N-hydroxysuccinimide is 1 to 2 times the mass of chondroitin sulfate; and the mass of the adipic acid dihydrazide is 0.67 to 2 times the mass of chondroitin sulfate.

[0013] In the above-mentioned method for preparing the injectable nanocomposite hydrogel for relieving osteoarthritis, in step (3), the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 to 2 times the mass of hyaluronic acid, the mass of the N-hydroxysuccinimide is 1 to 2 times the mass of hyaluronic acid, and the mass of the 3-aminophenylboronic acid is 0.2 to 1 times the mass of hyaluronic acid.

[0014] In the above-mentioned method for preparing the injectable nanocomposite hydrogel for relieving osteoarthritis, in step (4), the mass of the sodium periodate is 1 to 3 times the mass of the hyaluronic acid grafted with phenylboronic acid groups; the volume of the ethylene glycol is 1.25 to 6.25 times the mass of the hyaluronic acid grafted with phenylboronic acid groups, the volume unit of the ethylene glycol is mL, and the mass unit of the chondroitin sulfate is g.

[0015] In the method for preparing the injectable nanocomposite hydrogel for relieving osteoarthritis, in step (5), the concentration of the hydrophilic hydroxyapatite doped with strontium and europium ions is 0.05-1 g / mL, the concentration of the chondroitin sulfate solution grafted with hydrazide groups is 5-50%, the concentration of the oxidized hyaluronic acid solution grafted with phenylboronic acid groups is 5-50%, and the concentration of the recombinant collagen solution is 5-50%; the volume ratio of the chondroitin sulfate solution grafted with hydrazide groups, the oxidized hyaluronic acid solution grafted with phenylboronic acid groups, and the recombinant collagen solution is (1-5):(1-10):(1-5).

[0016] The injectable nanocomposite hydrogel prepared by the above method is used in the preparation of medicines for treating and relieving osteoarthritis.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. The hydrophilic hydroxyapatite doped with strontium and europium ions in the injectable nanocomposite hydrogel of the present invention is in the form of nanorods with a length of 150 to 300 nm and a diameter of 10 to 15 nm. The strontium ions have the ability to promote chondrocyte proliferation, and the europium ions have the ability to promote the polarization of macrophages from M1 to M2, thereby alleviating inflammation.

[0019] 2. The chondroitin sulfate, hyaluronic acid, and recombinant collagen in the injectable nanocomposite hydrogel of the present invention are all natural cartilage components, have good biocompatibility, and can simulate the extracellular matrix environment of cartilage cells to play a lubricating role;

[0020] 3. The hydrophilic hydroxyapatite doped with strontium and europium ions in the injectable nanocomposite hydrogel of the present invention is bonded to oxidized hyaluronic acid grafted with phenylboronic acid groups via a borate bond. The borate bond acts as a chemical bond that breaks when exposed to the high-reactive oxygen level microenvironment of osteoarthritis, thereby uniformly dispersing the hydrophilic hydroxyapatite doped with strontium and europium ions in the hydrogel and achieving responsive release.

[0021] 4. The injectable nanocomposite hydrogel of the present invention can be implanted into the osteoarthritis site via a non-invasive or minimally invasive approach, creating a favorable local immune microenvironment to immunomodulate macrophages to alleviate osteoarthritis and promote subchondral bone repair, while avoiding damage to surrounding normal tissues.

[0022] 5. The preparation method of the injectable nanocomposite hydrogel of the present invention is simple, the raw materials are cheap and easily available, the process is environmentally friendly, it is easy to scale up, and it has good market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 TEM image of the hydrophilic hydroxyapatite doped with strontium and europium ions of Example 1;

[0024] Figure 2 is the XRD pattern of the hydrophilic hydroxyapatite doped with strontium and europium ions of Example 1;

[0025] Figure 3 FT-IR images of the Sr and Eu co-doped hydroxyapatite before and after hydrophilic modification in Example 1;

[0026] Figure 4 FT-IR images of chondroitin sulfate before and after modification in Example 4;

[0027] Figure 5 1H-NMR diagram of chondroitin sulfate before and after modification in Example 4;

[0028] Figure 6 FT-IR images of hyaluronic acid before and after modification in Example 7;

[0029] Figure 7 1H-NMR diagrams of hyaluronic acid before and after modification in Example 7;

[0030] Figure 8 Schematic diagram of the injectable performance of the injectable hydrogel described in Example 10;

[0031] Figure 9 Schematic diagram of the experimental results of the efficacy of the injectable hydrogel on chondrocytes in Example 10. DETAILED DESCRIPTION

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified. The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and examples. Example 1

[0033] This embodiment provides a hydrophilic hydroxyapatite doped with strontium and europium ions, and the preparation method thereof includes:

[0034] (1) Under magnetic stirring, 1 mL of methyl oleate and 3 mL of methanol were mixed to obtain a precursor solution; 5.6 mL of a 0.28 M Ca(NO3)2 aqueous solution, 0.7 mL of a 0.28 M Sr(NO3)2 aqueous solution, and 0.7 mL of a 0.28 M Eu(NO3)3 aqueous solution were added to the precursor solution, stirred for 10 min, and 7 mL of a 0.168 M Na3PO4 aqueous solution was added and stirred for 10 min to obtain a mixed solution A; the mixed solution A was placed in a polytetrafluoroethylene reactor, sealed, and reacted at 130°C for 10 h; the system was cooled to room temperature after reaction, and the precipitate was collected by centrifugation;

[0035] (2) Disperse 0.05 g of precipitate in 100 mL of Tris-HCl buffer, add 0.15 g of tannic acid, stir for 4 h, and collect the precipitate by centrifugation with anhydrous ethanol to obtain hydrophilic hydroxyapatite doped with strontium and europium ions.

[0036] Figure 1 TEM image of hydrophilic hydroxyapatite doped with strontium and europium ions in Example 1. Figure 1 It can be seen that the hydrophilic hydroxyapatite doped with strontium and europium ions has a length of 150-300 nm and a width of 10-15 nm, and is in the shape of nanorods with uniform size.

[0037] Figure 2The XRD pattern of the hydrophilic hydroxyapatite doped with strontium and europium ions in Example 1. Figure 2 It can be seen that the comparison with the hydroxyapatite standard card JCPDS no.74-0565 shows that the product is hydroxyapatite.

[0038] Figure 3 FT-IR images of the Sr and Eu co-doped hydroxyapatite before and after hydrophilic modification in Example 1. Figure 3 Visible, 3500~3000 cm -1 The stretching vibration peak of the hydrophilic -OH group appeared, indicating that the hydrophilic modification was successful. Example 2

[0039] This embodiment provides a hydrophilic hydroxyapatite doped with strontium and europium ions, and the preparation method thereof includes:

[0040] (1) Under magnetic stirring, 1 mL of methyl oleate and 5 mL of methanol were mixed to obtain a precursor solution; 6.3 mL of a 0.28 M Ca(NO3)2 aqueous solution, 0.35 mL of a 0.28 M Sr(NO3)2 aqueous solution, and 0.35 mL of a 0.28 M Eu(NO3)3 aqueous solution were added to the precursor solution, and the mixture was stirred for 10 min. 7 mL of a 0.168 M Na3PO4 aqueous solution was added and stirred for 10 min to obtain a mixed solution A; the mixed solution A was placed in a polytetrafluoroethylene reactor, sealed, and reacted at 100 °C for 5 h; the reaction system was cooled to room temperature, and the precipitate was collected by centrifugation;

[0041] (2) Disperse 0.05 g of precipitate in 100 mL of Tris-HCl buffer, add 0.2 g of tannic acid, stir for 8 h, and collect the precipitate by centrifugation with anhydrous ethanol to obtain hydrophilic hydroxyapatite doped with strontium and europium ions.

[0042] The performance of the hydrophilic hydroxyapatite doped with strontium and europium ions in this embodiment is basically the same as that in Example 1. Example 3

[0043] This embodiment provides a hydrophilic hydroxyapatite doped with strontium and europium ions, and the preparation method thereof includes:

[0044] (1) Under magnetic stirring, 1 mL of methyl oleate and 7 mL of methanol were mixed to obtain a precursor solution; 4.9 mL of a 0.28 M Ca(NO3)2 aqueous solution, 1.05 mL of a 0.28 M Sr(NO3)2 aqueous solution, and 1.05 mL of a 0.28 M Eu(NO3)3 aqueous solution were added to the precursor solution, stirred for 10 min, and 7 mL of a 0.168 M Na3PO4 aqueous solution was added and stirred for 10 min to obtain a mixed solution A; the mixed solution A was placed in a polytetrafluoroethylene reactor, sealed, and reacted at 200°C for 15 h; the system was cooled to room temperature after reaction, and the precipitate was collected by centrifugation;

[0045] (2) Disperse 0.05 g of precipitate in 100 mL of Tris-HCl buffer, add 0.4 g of tannic acid, stir for 12 h, and collect the precipitate by centrifugation with anhydrous ethanol to obtain hydrophilic hydroxyapatite doped with strontium and europium ions.

[0046] The performance of the hydrophilic hydroxyapatite doped with strontium and europium ions in this embodiment is basically the same as that in Example 1. Example 4

[0047] This embodiment provides a solution of chondroitin sulfate CS-ADH containing a grafted hydrazide group, the preparation method of which comprises:

[0048] (2) Dissolve 1 g of chondroitin sulfate CS in 100 mL of deionized water under stirring, adjust the pH to 5-6 with 1 M hydrochloric acid solution, add 1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 2 g of N-hydroxysuccinimide, and stir for 10 min to activate the carboxyl groups.

[0049] (2) Add 0.67 g of adipic acid dihydrazide to the system after activation of the carboxyl group and stir at room temperature for 6 h;

[0050] (3) The reaction system was transferred to a dialysis bag with a cutoff of 3500Da and dialyzed with deionized water for 3 days. The deionized water was replaced every day during the dialysis process. The dialyzed system was freeze-dried to obtain chondroitin sulfate CS-ADH grafted with hydrazide groups;

[0051] (4) Dissolve 0.05 g of CS-ADH in 1 mL of PBS to obtain a solution of chondroitin sulfate CS-ADH containing grafted hydrazide groups.

[0052] Figure 4 FT-IR images of chondroitin sulfate before and after modification in Example 4. Figure 4 Visible, 1635 cm -1 A characteristic absorption peak attributed to the amino group appears at , indicating that the grafting of the hydrazide group is successful.

[0053] Figure 5 Example 4 1H-NMR diagram of chondroitin sulfate before and after modification. Figure 5 It can be seen that compared with CS, characteristic absorption peaks appeared at 1.5 and 2.2 ppm in the CS-ADH curve, indicating that the grafting of hydrazide groups was successful. Example 5

[0054] This embodiment provides a solution of chondroitin sulfate CS-ADH containing a grafted hydrazide group, the preparation method of which comprises:

[0055] (1) Dissolve 10 g of chondroitin sulfate CS in 100 mL of deionized water under stirring. Adjust the pH of the solution to 5-6 with 1 M hydrochloric acid solution. Add 20 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 10 g of N-hydroxysuccinimide and stir for 30 min to activate the carboxyl groups.

[0056] (2) Add 20 g of adipic acid dihydrazide to the system after activation of the carboxyl group and stir at room temperature for 15 h;

[0057] (3) The reaction system was transferred to a dialysis bag with a cutoff of 3500Da and dialyzed with deionized water for 5 days. The deionized water was replaced every day during the dialysis process. The dialyzed system was freeze-dried to obtain chondroitin sulfate grafted with hydrazide groups CS-ADH; the performance of the chondroitin sulfate grafted with hydrazide groups in this example was basically the same as that in Example 4;

[0058] (4) Dissolve 0.2 g of CS-ADH in 1 mL of PBS to obtain a solution of chondroitin sulfate CS-ADH containing grafted hydrazide groups. Example 6

[0059] This embodiment provides a solution of chondroitin sulfate CS-ADH containing a grafted hydrazide group, the preparation method of which comprises:

[0060] (1) Dissolve 10 g of chondroitin sulfate CS in 500 mL of deionized water under stirring. Adjust the pH of the solution to 5-6 with 1 M hydrochloric acid solution. Add 15 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 15 g of N-hydroxysuccinimide and stir for 50 min to activate the carboxyl groups.

[0061] (2) Add 15 g of adipic acid dihydrazide to the system after activation of the carboxyl group and stir at room temperature for 24 h;

[0062] (3) The reaction system was transferred to a dialysis bag with a cutoff of 3500 Da and dialyzed with deionized water for 4 days. The deionized water was replaced every day during the dialysis process. The dialyzed system was freeze-dried to obtain chondroitin sulfate grafted with hydrazide groups CS-ADH. The performance of the chondroitin sulfate grafted with hydrazide groups in this example was basically the same as that in Example 4.

[0063] (4) Dissolve 0.5 g of CS-ADH in 1 mL of PBS to obtain a solution of chondroitin sulfate CS-ADH containing grafted hydrazide groups. Example 7

[0064] This embodiment provides a solution of oxidized hyaluronic acid (OHP) containing grafted phenylboronic acid groups. The oxidized hyaluronic acid with grafted phenylboronic acid groups is obtained by first grafting 3-aminophenylboronic acid onto hyaluronic acid and then performing formaldehyde modification. The preparation method includes:

[0065] (1) Under stirring conditions, 1 g of hyaluronic acid was dissolved in 100 mL of deionized water, and the pH was adjusted to 5-6 with 1 M hydrochloric acid solution. 2 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1 g of N-hydroxysuccinimide were added and stirred for 10 min to activate the carboxyl groups. 0.4 g of 3-aminophenylboronic acid was added to the system after carboxyl group activation, and the reaction was stirred at room temperature for 6 h. The reaction system was transferred to a dialysis bag with a cutoff of 3500 Da and dialyzed with deionized water for 3 days to obtain hyaluronic acid (HP) grafted with phenylboronic acid groups.

[0066] (2) Under stirring conditions, 0.8 g of hyaluronic acid grafted with phenylboronic acid groups was dissolved in 80 mL of deionized water, 0.8 g of sodium periodate was added, and the mixture was stirred in the dark for 1 h. 5 mL of ethylene glycol was added to the reaction system, and the reaction was terminated by stirring for 1 h. The terminated reaction system was transferred to a dialysis bag with a cutoff of 3500 Da, and dialyzed with deionized water for 3 days. The deionized water was replaced every day during the dialysis process. The dialyzed system was freeze-dried to obtain oxidized hyaluronic acid grafted with phenylboronic acid groups (OHP).

[0067] (3) Dissolve 0.05 g of OHP in 1 mL of PBS to obtain a solution of hyaluronic acid OHP containing grafted phenylboronic acid groups.

[0068] Figure 6 The FT-IR images of Example 7 before and after hyaluronic acid modification are shown. Figure 6 It can be seen that compared with HA, the 1558 cm -1 The characteristic absorption peak at 1645 cm-1 in the OHP curve is attributed to the phenylboronic acid group, indicating that the grafting of the phenylboronic acid group is successful and the modification of HP is successful. -1The appearance of characteristic peaks belonging to aldehyde groups indicated that the modification of OHP was successful.

[0069] Figure 7 1H-NMR diagram of hyaluronic acid before and after modification in Example 7. Figure 7 It can be seen that compared with HA, a new characteristic peak appears at 7.4~7.8 ppm in the HP curve, indicating that the modification of HP is successful; compared with HP, a new characteristic peak appears at 4.9~5.0 ppm in the OHP curve, indicating that the modification of OHP is successful. Example 8

[0070] This embodiment provides a solution of oxidized hyaluronic acid (OHP) containing grafted phenylboronic acid groups, the preparation method of which includes:

[0071] (1) Under stirring conditions, 8 g of hyaluronic acid was dissolved in 800 mL of deionized water, and the pH was adjusted to 5-6 with 1 M hydrochloric acid solution. 16 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 16 g of N-hydroxysuccinimide were added and stirred for 30 min to activate the carboxyl groups. 5 g of 3-aminophenylboronic acid was added to the system after carboxyl group activation, and the reaction was stirred at room temperature for 15 h. The reaction system was transferred to a dialysis bag with a cutoff of 3500 Da and dialyzed with deionized water for 5 days to obtain hyaluronic acid grafted with phenylboronic acid groups (HP).

[0072] (2) Under stirring conditions, 8 g of hyaluronic acid grafted with phenylboronic acid groups was dissolved in 800 mL of deionized water, 24 g of sodium periodate was added, and the mixture was stirred in the dark for 3 h; 10 mL of ethylene glycol was added to the reaction system, and the reaction was terminated by stirring for 3 h; the terminated reaction system was transferred to a dialysis bag with a cutoff of 3500 Da, and dialyzed with deionized water for 5 days. The deionized water was replaced every day during the dialysis process, and the dialyzed system was freeze-dried to obtain oxidized hyaluronic acid grafted with phenylboronic acid groups (OHP);

[0073] (3) Dissolve 0.2 g of OHP in 1 mL of PBS to obtain a solution of hyaluronic acid OHP containing grafted phenylboronic acid groups. Example 9

[0074] This embodiment provides a solution of oxidized hyaluronic acid (OHP) containing grafted phenylboronic acid groups, the preparation method of which includes:

[0075] (1) Dissolve 10 g of hyaluronic acid in 500 mL of deionized water under stirring, adjust the pH to 5-6 with 1 M hydrochloric acid solution, add 15 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 15 g of N-hydroxysuccinimide, and stir for 50 min to activate the carboxyl groups; add 7 g of 3-aminophenylboronic acid to the system after carboxyl group activation, and stir at room temperature for 24 h; transfer the reaction system to a dialysis bag with a cutoff of 3500 Da, and dialyze with deionized water for 4 days to obtain hyaluronic acid (HP) grafted with phenylboronic acid groups;

[0076] (2) Under stirring conditions, 2 g of hyaluronic acid grafted with phenylboronic acid groups was dissolved in 80 mL of deionized water, 4 g of sodium periodate was added, and the mixture was stirred in the dark for 5 h. 8 mL of ethylene glycol was added to the reaction system, and the reaction was terminated by stirring for 5 h. The terminated reaction system was transferred to a dialysis bag with a cutoff of 3500 Da, and dialyzed with deionized water for 4 days. The deionized water was replaced every day during the dialysis process. The dialyzed system was freeze-dried to obtain oxidized hyaluronic acid grafted with phenylboronic acid groups (OHP).

[0077] (3) Dissolve 0.5 g of OHP in 1 mL of PBS to obtain a solution of hyaluronic acid OHP containing grafted phenylboronic acid groups. Example 10

[0078] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0079] 0.05 g of hydrophilic hydroxyapatite doped with strontium and europium ions, 0.33 mL of a 5% solution of chondroitin sulfate grafted with hydrazide groups, 0.33 mL of a 5% solution of oxidized hyaluronic acid grafted with phenylboronic acid groups, and 0.33 mL of a 5% solution of recombinant collagen were mixed and stirred to obtain an injectable nanocomposite hydrogel.

[0080] Injectable hydrogel performance evaluation:

[0081] Figure 8 Schematic diagram of the injectable performance of the injectable hydrogel described in Example 10. The injectable hydrogel was loaded into a syringe and injected into the target site. The results showed that the hydrogel of the present invention can be injected.

[0082] Cytology experiments

[0083] The injectable hydrogel described in Example 10 was used as a research object to investigate its efficacy in promoting the growth of normal cells (chondrocytes), specifically including:

[0084] (1) Preparation of DMEM extract of injectable hydrogel

[0085] The injectable hydrogel was freeze-dried, sterilized by Co-60 irradiation, and immersed in DMEM complete culture medium at a concentration of 0.1 g / mL for 3 days (37° C.) to obtain a hydrogel extract, which was filtered with a filter membrane before use to obtain a DMEM extract of the injectable hydrogel;

[0086] (2) Determination of chondrocyte toxicity of injectable hydrogels:

[0087] ①Cell layout: 1×10 4 The cell dispersion was placed in a well plate at a cell density of 100 μL per well of a 96-well plate and cultured in an incubator (37°C, 5% CO2) for 24 h.

[0088] ② Cytotoxicity detection: The 96-well plates where the chondrocytes had attached were removed separately, and the culture medium was replaced with the DMEM extract of the injectable hydrogel in step 1. The cells were cultured in an incubator (37°C, 5% CO2). At 24 and 48 hours of culture, 50 μL of MTT was added to each well, and the cells were incubated for another 2-4 hours. After that, all the liquid in the wells was removed, 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 the cell viability was calculated based on the OD value. The blank group was cultured with complete DMEM medium.

[0089] The results of the hydrogel's efficacy on chondrocytes are as follows Figure 9 As shown. Figure 9 It can be seen that after culturing for 24 hours and 48 hours, the survival rate of chondrocytes was higher than 100%, indicating that the hydrogel had no cytotoxicity to chondrocytes and had the effect of promoting cartilage proliferation and accelerating the repair of cartilage defects. Example 11

[0090] This embodiment provides an injectable hydrogel, the preparation method of which includes:

[0091] 0.5 g of hydrophilic hydroxyapatite doped with strontium and europium ions, 0.3 mL of a 30% solution of chondroitin sulfate grafted with hydrazide groups, 0.5 mL of a 30% solution of oxidized hyaluronic acid grafted with phenylboronic acid groups, and 0.2 mL of a 30% solution of recombinant collagen were mixed and stirred to obtain an injectable nanocomposite hydrogel.

[0092] The performance of the injectable hydrogel in this example is basically the same as that in Example 10. Example 12

[0093] 1 g of hydrophilic hydroxyapatite doped with strontium and europium ions, 0.3125 mL of a 30% solution of chondroitin sulfate grafted with hydrazide groups, 0.625 mL of a 30% solution of oxidized hyaluronic acid grafted with phenylboronic acid groups, and 0.0625 mL of a 30% solution of recombinant collagen were mixed and stirred to obtain an injectable nanocomposite hydrogel.

[0094] The performance of the injectable hydrogel in this example is basically the same as that in Example 10.

[0095] The above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Any modifications, alterations, or equivalent structural variations to the above embodiments based on the essence of the present invention remain within the scope of protection of the present invention. This research was supported by the National Key R&D Program of China (Design, Construction, and Pathway Assembly of Key Enzyme Elements for Glycosaminoglycan Derivatives and Glycoprotein Synthesis, 2023YFA0914301).

Claims

1. A method for preparing an injectable nanocomposite hydrogel for relieving osteoarthritis, characterized in that The following steps are involved: (1) preparing hydrophilic hydroxyapatite doped with strontium and europium ions, specifically comprising: dissolving aqueous solutions of Ca(NO3)2, Sr(NO3)2, and Eu(NO3)3 and an aqueous solution of Na3PO4 in a methanol solution of methyl oleate in sequence to obtain a mixed solution, reacting the mixed solution in a closed reactor at 100-200°C for 5-15 hours, and collecting a precipitate; dispersing the precipitate in a Tris-HCl buffer, adding tannic acid, stirring thoroughly, and collecting the precipitate to obtain hydrophilic hydroxyapatite doped with strontium and europium ions; (2) Preparing a chondroitin sulfate solution grafted with a hydrazide group, specifically comprising: dissolving 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in an aqueous solution of chondroitin sulfate at a pH of 5 to 6 and stirring thoroughly to activate the carboxyl group; adding adipic acid dihydrazide to the system after the carboxyl group activation to fully react; transferring the system after the reaction to dialysis, freeze-drying, and obtaining a chondroitin sulfate grafted with a hydrazide group; dissolving the chondroitin sulfate grafted with a hydrazide group in a PBS buffer solution to obtain a chondroitin sulfate solution containing a grafted hydrazide group; (3) Preparing a hyaluronic acid solution grafted with phenylboronic acid groups, specifically comprising: placing 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide in an aqueous solution of hyaluronic acid at a pH of 5 to 6 and stirring thoroughly to activate the carboxyl groups; adding 3-aminophenylboronic acid to the system after the carboxyl groups are activated and reacting thoroughly; transferring the system after the reaction to dialyze, and freeze-drying to obtain hyaluronic acid grafted with phenylboronic acid groups; dissolving the oxidized hyaluronic acid grafted with phenylboronic acid groups in deionized water to obtain an aqueous solution of hyaluronic acid containing grafted phenylboronic acid groups; (4) preparing a solution of oxidized hyaluronic acid grafted with phenylboronic acid groups, specifically comprising: dissolving sodium periodate in an aqueous solution of hyaluronic acid grafted with phenylboronic acid groups, adding ethylene glycol after sufficient reaction in the dark, and stirring to terminate the reaction; dialyzing the system after the termination of the reaction, and freeze-drying to obtain oxidized hyaluronic acid grafted with phenylboronic acid groups; dissolving the oxidized hyaluronic acid grafted with phenylboronic acid groups in a PBS buffer solution to obtain an oxidized hyaluronic acid solution containing grafted phenylboronic acid groups; (5) Preparing an injectable nanocomposite hydrogel, specifically comprising: uniformly blending the hydrophilic hydroxyapatite doped with strontium and europium ions in step (1), the chondroitin sulfate solution grafted with hydrazide groups in step (2), the oxidized hyaluronic acid solution grafted with phenylboronic acid groups in step (4), and the recombinant collagen solution to obtain the injectable nanocomposite hydrogel.

2. The method for preparing the injectable nanocomposite hydrogel according to claim 1, wherein: In step (1), in the methanol solution of methyl oleate, the volume of methanol is 3 to 7 times the volume of methyl oleate; the molar ratio of (Ca+Sr+Eu) / P is 1.67, the molar ratio of Sr / (Ca+Sr+Eu) is 0.05 to 0.5; the molar ratio of Eu / (Ca+Sr+Eu) is 0.05 to 0.5; and the mass of the tannic acid is 3 to 6 times the mass of the precipitate.

3. The method for preparing the injectable nanocomposite hydrogel according to claim 1, wherein: In step (2), the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 to 2 times the mass of chondroitin sulfate, the mass of the N-hydroxysuccinimide is 1 to 2 times the mass of chondroitin sulfate; and the mass of the adipic acid dihydrazide is 0.67 to 2 times the mass of chondroitin sulfate.

4. The method for preparing the injectable nanocomposite hydrogel according to claim 1, wherein: In step (3), the mass of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide is 1 to 2 times the mass of the hyaluronic acid, the mass of the N-hydroxysuccinimide is 1 to 2 times the mass of the hyaluronic acid, and the mass of the 3-aminophenylboronic acid is 0.2 to 1 times the mass of the hyaluronic acid.

5. The method for preparing the injectable nanocomposite hydrogel according to claim 1, wherein: In step (4), the mass of the sodium periodate is 1 to 3 times the mass of the hyaluronic acid grafted with phenylboronic acid groups; and the volume of the ethylene glycol is 1.25 to 6.25 times the mass of the hyaluronic acid grafted with phenylboronic acid groups.

6. The method for preparing the injectable nanocomposite hydrogel according to claim 1, wherein: In step (5), the concentration of the hydrophilic hydroxyapatite doped with strontium and europium ions is 0.05-1 g / mL, the concentration of the chondroitin sulfate solution grafted with hydrazide groups is 5-50%, the concentration of the oxidized hyaluronic acid solution grafted with phenylboronic acid groups is 5-50%, and the concentration of the recombinant collagen solution is 5-50%; the volume ratio of the chondroitin sulfate solution grafted with hydrazide groups, the oxidized hyaluronic acid solution grafted with phenylboronic acid groups, and the recombinant collagen solution is (1-5):(1-10):(1-5).

7. The injectable nanocomposite hydrogel prepared by the method according to any one of claims 1 to 6.

8. Use of the injectable nanocomposite hydrogel according to claim 7 in the preparation of medicines for treating and relieving osteoarthritis.

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