A modified hyaluronic acid hydrogel and a method for preparing the same

Abstract

The present disclosure provides a hyaluronic acid-based hydrogel and a preparation method and use thereof. The modified hyaluronic acid hydrogel of the present disclosure is formed by tyramine-modified hyaluronic acid and poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid under the action of horseradish peroxidase and hydrogen peroxide. The crosslinking agent used in the preparation of the modified hyaluronic acid hydrogel of the present disclosure is horseradish peroxidase, and the prepared hydrogel has good biocompatibility; the hydrogel contains a component with a brush structure, and the prepared hydrogel has good lubricity, which is conducive to the treatment of osteoarthritis.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to biocompatible materials, in particular to a modified hyaluronic acid hydrogel capable of loading bioactive substances and a preparation method thereof. BACKGROUND

[0002] Osteoarthritis, as the most common type of arthritis in clinic, is a degenerative joint disease. Its pathogenesis is mainly due to the secretion of pro-inflammatory factors by lymphocytes and macrophages in synovial tissue, which gradually affects cartilage, synovial membrane and bone tissue, and eventually leads to chronic inflammation and joint dysfunction. At present, the commonly used strategies for repairing articular cartilage in clinic include non-steroidal anti-inflammatory drug treatment, microfracture surgery treatment, autologous cell microinjection treatment and total knee arthroplasty, etc. However, each of the above treatment methods has its own limitations, including drug side effects, slow tissue regeneration, surgical trauma, infection and prosthesis stiffness, etc. Therefore, it is of great research value to develop new strategies for treating osteoarthritis.

[0003] In recent years, the development of tissue engineering and regenerative medicine has brought new ideas for the treatment of osteoarthritis. Hyaluronic acid (HA) is a linear mucopolysaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine as disaccharide units connected by alternating β-l, 3 and β-l, 4 glycosidic chains, which exists widely in human tissues. Hyaluronic acid has good biocompatibility, biodegradability, viscoelasticity and easy modification, etc., and is therefore considered to be very suitable for developing new bioactive scaffolds for the treatment of osteoarthritis. However, the preparation of hyaluronic acid biological scaffolds usually involves the use of chemical reagents with obvious cytotoxicity, and the limited lubricity and anti-inflammatory activity limit the further application of this strategy.

[0004] Exosomes are small vesicles with phospholipid bilayer structure produced by cell paracrine. In recent years, they have been widely concerned by researchers due to their good biological activity in various diseases. Many studies have shown that mesenchymal stem cell-derived exosomes have good anti-inflammatory activity and are the most widely used exosomes by researchers. However, the half-life of exosomes in vivo is usually short, which limits their further application. SUMMARY

[0005] In view of the problems of existing hyaluronic acid biological scaffolds, such as unsatisfactory biocompatibility and safety, insufficient lubricity and anti-inflammatory activity, the present disclosure provides a modified hyaluronic acid-based hydrogel, a preparation method thereof and uses thereof.

[0006] An aspect of the present disclosure provides a modified hyaluronic acid hydrogel formed from tyramine-modified hyaluronic acid and poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid under the action of horseradish peroxidase and hydrogen peroxide.

[0007] The present disclosure further provides a modified hyaluronic acid hydrogel loaded with a bioactive substance formed from tyramine-modified hyaluronic acid, poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid, and a bioactive substance under the action of horseradish peroxidase and hydrogen peroxide.

[0008] In a further embodiment of the present disclosure, the bioactive substance is one or more selected from mesenchymal stem cell-derived exosomes, curcumin, quercetin, and ganoderma polysaccharide.

[0009] Another aspect of the present disclosure provides a method for preparing a modified hyaluronic acid hydrogel, comprising the following steps:

[0010] tyramine-modified hyaluronic acid is prepared into an aqueous solution;

[0011] poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared into an aqueous solution;

[0012] The aqueous solution of tyramine-modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid are mixed, horseradish peroxidase and hydrogen peroxide are added to the mixture, and vortexing is performed to obtain a modified hyaluronic acid hydrogel.

[0013] In a further embodiment of the present disclosure, the tyramine-modified hyaluronic acid is prepared by the following steps:

[0014] Hyaluronic acid is dissolved in a buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, stirring is performed, tyramine is added, and stirring is performed for reaction;

[0015] After the reaction is completed, the reaction product is dialyzed, and lyophilization is performed to obtain tyramine-modified hyaluronic acid.

[0016] In a further embodiment of the present disclosure, the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared by the following steps:

[0017] Hyaluronic acid is dissolved in deionized water, adipic acid dihydrazide is added, anhydrous ethanol is added, and the pH is adjusted as the reaction progresses;

[0018] After the reaction is completed, the reaction product is dialyzed, and lyophilization is performed to obtain adipic acid dihydrazide-modified hyaluronic acid;

[0019] The hyaluronic acid modified by adipic acid dihydrazide and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are prepared into aqueous solutions, mixed, the pH is adjusted, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the pH is adjusted, and the reaction is stirred;

[0020] After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the hyaluronic acid modified by poly-2-acrylamide-2-methylpropanesulfonic acid sodium is prepared.

[0021] Another aspect of the present disclosure provides a method for preparing a modified hyaluronic acid hydrogel loaded with a bioactive substance, comprising the following steps:

[0022] The hyaluronic acid modified by tyramine is prepared into an aqueous solution;

[0023] The hyaluronic acid modified by poly-2-acrylamide-2-methylpropanesulfonic acid sodium is prepared into an aqueous solution;

[0024] The aqueous solution of hyaluronic acid modified by tyramine, the aqueous solution of hyaluronic acid modified by poly-2-acrylamide-2-methylpropanesulfonic acid sodium, and the bioactive substance are mixed, horseradish peroxidase and hydrogen peroxide are added to the mixed solution, and vortexed to obtain a modified hyaluronic acid hydrogel loaded with a bioactive substance.

[0025] Another aspect of the present disclosure provides a hydrogel precursor composition comprising an aqueous solution of hyaluronic acid modified by tyramine and hyaluronic acid modified by poly-2-acrylamide-2-methylpropanesulfonic acid sodium.

[0026] Another aspect of the present disclosure provides a kit comprising an aqueous solution of hyaluronic acid modified by tyramine, hyaluronic acid modified by poly-2-acrylamide-2-methylpropanesulfonic acid sodium, horseradish peroxidase, hydrogen peroxide, and deionized water.

[0027] Still another aspect of the present disclosure provides the use of a modified hyaluronic acid hydrogel in the preparation of a preparation, a drug, or a medical device for treating osteoarthritis.

[0028] The technical solution of the present disclosure has the following advantages:

[0029] In the preparation of the modified hyaluronic acid hydrogel of the present disclosure, a traditional chemical crosslinking agent with cytotoxicity is not needed, and horseradish peroxidase with good biocompatibility is used to promote the formation of the hydrogel, and the prepared hydrogel has good biocompatibility and safety.

[0030] The poly-2-acrylamide-2-methylpropane sulfonic acid sodium modified hyaluronic acid can form a new brush structure in the molecular structure, which is beneficial to increase the lubricity of the hydrogel prepared therefrom, so that the modified hyaluronic acid hydrogel of the present disclosure can be applied to the joint and the like for the treatment of osteoarthritis.

[0031] The modified hyaluronic acid hydrogel of the present disclosure can be flexibly adjusted in mechanical strength to adapt to different application scenarios. The modified hyaluronic acid hydrogel of the present disclosure also has good injectability and tissue adhesion, and is easy to be prepared into a preparation suitable for injection for local use in joints and the like; the hydrogel can be attached to the applied site to play a role of lubrication, buffering and the like.

[0032] The modified hyaluronic acid hydrogel of the present disclosure can also load bioactive substances, such as mesenchymal stem cell-derived exosomes. Under the protection of the modified hyaluronic acid hydrogel, the half-life of the active substances is prolonged, and the biological effects thereof can be stably and persistently exerted locally in the joint, thereby improving the treatment effect on osteoarthritis and the like. BRIEF DESCRIPTION OF DRAWINGS

[0033] The present disclosure is described in detail according to one or more different embodiments with reference to the following drawings. The provided drawings are for the purpose of facilitating the understanding of the present disclosure, and should not be considered as a limitation on the breadth, scope or applicability of the present disclosure.

[0034] Figure 1 is the FTIR detection spectrum of HA, HA-Tyr, HA-ADH and HA-PA.

[0035] Figure 2 is the H-NMR detection spectrum of HA and HA-PA.

[0036] Figure 3 is the process of forming a hydrogel from a HA-Tyr / HA-PA modified hydrogel precursor solution.

[0037] Figure 4 is the rheological analysis result graph of the modified hyaluronic acid hydrogel.

[0038] Figure 5 is the phenomenon of adding HRP and hydrogen peroxide to the mixed solution of HA-Tyr and HA-DA.

[0039] Figure 6 is the case of the modified hyaluronic acid hydrogel passing through a syringe needle.

[0040] Figure 7 is the tissue adhesion test result of the modified hyaluronic acid hydrogel.

[0041] Figure 8 is the atomic force microscope detection graph of the modified hyaluronic acid hydrogel.

[0042] Figure 9 are the process of forming hydrogels from HA-Tyr / HA-PA modified hydrogel precursor solutions with different ratios.

[0043] Figure 10 are the SEM detection images of the hydrogels formed from HA-Tyr / HA-PA modified hydrogel precursor solutions with different ratios. DETAILED DESCRIPTION

[0044] One aspect of the present disclosure relates to a modified hyaluronic acid hydrogel and a method for preparing the same.

[0045] The modified hyaluronic acid hydrogel of the present disclosure is formed by tyramine (Tyr) modified hyaluronic acid (HA-Tyr) and poly-(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS) modified hyaluronic acid (HA-PA) together.

[0046] Tyramine modified hyaluronic acid

[0047] One of the components for forming the modified hyaluronic acid hydrogel of the present disclosure is tyramine modified hyaluronic acid. After hyaluronic acid is modified by tyramine, phenolic hydroxyl groups are present in the molecule, which can be used for subsequent gel formation.

[0048] From the perspective of application in joints and other parts, the molecular weight of the raw material hyaluronic acid before modification is preferably 1300-1500 kDa.

[0049] Hyaluronic acid is subjected to amidation reaction with tyramine to form tyramine modified hyaluronic acid, which can be carried out under the action of activating agents N-hydroxysuccinimide (NHS) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC). The basic reaction process is as follows:

[0050]

[0051] An exemplary preparation method of tyramine modified hyaluronic acid is as follows:

[0052] A1) Dissolve hyaluronic acid in a buffer solution;

[0053] A2) Add NHS and EDC to the solution of A1), and stir well;

[0054] A3) Add tyramine to the solution of A2), and stir to react;

[0055] A4) Dialyze the reaction product of A3) to remove unreacted reagents;

[0056] A5) Freeze-drying the product solution of A4) to obtain HA-Tyr. It can be stored at 4°C.

[0057] wherein:

[0058] The buffer solution of step A1) can be 2-(N-morpholino)ethanesulfonic acid (MES) buffer solution, for example, MES buffer solution with a concentration of 0.01 M, pH = 5.5; the concentration of the prepared hyaluronic acid solution is 0.5-3 w / v%.

[0059] In step A2), the ratio of NHS to EDC can be 1:5-2:1.

[0060] In step A3), the amount of tyramine added can be 0.5-2 w / v%.

[0061] In step A4), the specific method of dialysis is not limited, and preferably, first dialysis is performed using a 0.5-1% sodium chloride solution, and then dialysis is performed using deionized water. A dialysis membrane / dialysis tube with a molecular weight cut-off (MWCO) of 3-20 kDa can be used.

[0062] Poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid

[0063] Yet another component of the modified hyaluronic acid hydrogel of the present disclosure is poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid.

[0064] From the perspective of application to joints and the like, the molecular weight of the raw material hyaluronic acid before modification is preferably 1300-1500 kDa.

[0065] The hyaluronic acid can be modified with adipic acid dihydrazide (ADH) first to obtain adipic acid dihydrazide-modified hyaluronic acid (HA-ADH) having a hydrazide group. Then, the hydrazide group of HA-ADH is further reacted with the terminal carboxyl group of PAMPS under the action of an activating agent EDC / NHS to obtain PAMPS-modified hyaluronic acid (HA-PA). The basic reaction process is as follows:

[0066] An exemplary preparation method of PAMPS-modified hyaluronic acid is as follows:

[0067] B1) Add HA to an appropriate amount of deionized water and stir until completely dissolved;

[0068] B2) Add the ADH solution to the solution of B1) and stir uniformly;

[0069] B3) Adjust the pH of the solution of B2) to 4-5, add anhydrous ethanol, stir, and then gradually adjust the pH until the reaction is complete;

[0070] B4) dialysis of the reaction product of B3) to remove unreacted reagents;

[0071] B5) lyophilization of the product solution of B4) to obtain HA-ADH. The HA-ADH can be stored at 4°C.

[0072] B6) separately dissolve HA-ADH and PAMPS into aqueous solutions, then mix the HA-ADH solution and the PAMPS solution, adjust the pH of the mixture to 4-5, and stir thoroughly;

[0073] B7) add EDC to the mixture obtained in B6), adjust the pH of the solution to 4-5, and stir to react;

[0074] B8) add NHS to the solution of B7), adjust the pH of the solution to 4-6, stir to react, and then adjust the pH of the reacted solution to 6.8-7.4;

[0075] B9) dialysis of the reaction product of B8) to remove unreacted reagents;

[0076] B10) lyophilization of the product solution of B9) to obtain HA-PA. The HA-PA can be stored at 4°C.

[0077] wherein:

[0078] The concentration of the HA solution prepared in step B1) is 0.2-1 w / v%.

[0079] The amount of ADH added in step B2) is 3-15 w / v%.

[0080] In step B3), the volume ratio of the amount of anhydrous ethanol added to the volume of the original solution is preferably 1:1; after stirring after the addition of anhydrous ethanol, the pH of the solution is adjusted to 4-5, stirred, and then the pH of the solution is adjusted to 6.8-7.4.

[0081] In step B4), dialysis is preferably performed sequentially using 100 mM sodium chloride solution, ethanol, and deionized water; a dialysis membrane / tube with a MWCO of 3-20 kDa can be selected.

[0082] In step B5), the grafting ratio of the prepared HA-ADH is preferably 70%-85%. The grafting ratio of HA-ADH can be calculated from the elemental analysis results of the product. The molecular weight of HA monomer is 403.31, and the grafting ratio of HA-ADH can be calculated according to the number of nitrogen atoms in the molecules of HA and ADH according to the following formula:

[0083] HA-ADH grafting ratio (%) = percentage of N content in HA-ADH product / (14 x 4 / 403.31)

[0084] In step B6), the concentration of the prepared HA-ADH solution is 0.2-1 w / v%; the concentration of the prepared PAMPS solution is 2-10 wt%, and the pH value is 2.0-3.0. The mixing ratio of the HA-ADH solution and the PAMPS solution is 1:1-1:3.

[0085] In step B7), the concentration of EDC is 0.5-1 w / v%.

[0086] In step B8), the concentration of NHS is 0.2-1 w / v%.

[0087] In step B9), it is preferred to perform dialysis with 100 mM sodium chloride solution, ethanol and deionized water in sequence; a dialysis membrane / tube with MWCO of 3-20 kDa can be optionally used.

[0088] Modified hyaluronic acid hydrogel

[0089] The HA-Tyr and HA-PA can form a modified hyaluronic acid hydrogel under appropriate catalysis. The HA-Tyr contains phenolic hydroxyl groups, which can undergo radical polymerization under the action of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). The HA-PA also participates in the formation of the spatial network of the modified hyaluronic acid hydrogel.

[0090] An exemplary preparation method of the modified hyaluronic acid hydrogel is as follows:

[0091] C1) Prepare the HA-Tyr and HA-PA into HA-Tyr solution and HA-PA solution with deionized water, respectively;

[0092] C2) Mix the prepared HA-Tyr solution and HA-PA solution in a certain ratio;

[0093] C3) Add HRP and hydrogen peroxide to the solution of C2), vortex, and form a modified hyaluronic acid hydrogel.

[0094] Wherein:

[0095] In step C1), the concentration of the prepared HA-Tyr solution is 0.2-3 w / v%, and the concentration of the prepared HA-PA solution is 0.05-2 w / v%;

[0096] In step C2), the mixing ratio of the HA-Tyr solution and the HA-PA solution is 5:1-1:5; if it is necessary to adjust the final concentration of HA-Tyr or HA-PA in the mixed solution, a certain amount of deionized water can be added according to the actual need in this step;

[0097] In step C3), the concentration of HRP is 0.5-10 U / ml, and the concentration of hydrogen peroxide is 0.1-5 mM, wherein HRP and hydrogen peroxide are added in a ratio of 50-500 μl per 1 ml of the mixed solution of C2).

[0098] Modified hyaluronic acid hydrogel loaded with active substances

[0099] The modified hyaluronic acid hydrogel of the present disclosure can be further loaded with active substances. A representative active substance is an exosome, particularly a mesenchymal stem cell-derived exosome. Other examples of active substances are small molecules with anti-inflammatory activity extracted from natural products, such as curcumin, quercetin, ganoderma polysaccharide, etc.

[0100] The desired active substance can be added to the mixed solution of HA-Tyr and HA-PA in step C2) of the aforementioned preparation of the modified hyaluronic acid hydrogel, followed by the subsequent HRP-hydrogen peroxide catalyzed crosslinking step. The modified hyaluronic acid hydrogel thus prepared is uniformly and stably loaded with the desired active substance.

[0101] Hydrogel precursor composition

[0102] The present disclosure also provides a hydrogel precursor composition comprising lyophilized HA-Tyr and lyophilized HA-PA. In use, the HA-Tyr and HA-PA can be dissolved in deionized water as needed, mixed, and then added with HRP and H2O2 to form a gel.

[0103] The present disclosure also provides a hydrogel preparation kit for ease of use, comprising lyophilized HA-Tyr, lyophilized HA-PA, HRP and H2O2, and can further comprise packaged deionized water. The modified hyaluronic acid hydrogel can be conveniently prepared using the kit. When using the kit, appropriate biologically active substances can also be added as needed.

[0104] The technical solutions of the present disclosure are further explained below in conjunction with examples. Those skilled in the art will understand that the following examples are only for illustration of the present disclosure and should not be considered as limiting the scope of the present disclosure. If no specific conditions are specified in the examples, the operations are carried out under conventional conditions or according to the manufacturer's recommendations. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0105] Example 1 Preparation of HA-Tyr

[0106] a) HA was added to a MES buffer solution with a concentration of 0.01 M and pH = 5.5, and stirred until completely dissolved to prepare a 1 w / v% HA solution;

[0107] b) NHS and EDC (in a ratio of 5:8) were added to the solution of a), and stirred at room temperature for 1 hour;

[0108] c) To the solution of b), add tyramine (1.03 w / v% of added amount), stir the reaction at room temperature for 72 hours;

[0109] d) Dialyze the reaction product of c) first with 0.6% sodium chloride solution for 24 hours, and then with deionized water for 48 hours, using a dialysis tube with MWCO = 3.5 kDa;

[0110] e) Freeze dry the solution of d) and store it at 4°C, which is HA-Tyr.

[0111] The Fourier transform infrared spectroscopy (FTIR) detection results of HA before modification and the prepared HA-Tyr are shown in Figure 1 .

[0112] Example 2 Preparation of HA-PA

[0113] a) Add HA into deionized water and stir until completely dissolved to prepare a HA solution with a concentration of 0.5 w / v%;

[0114] b) Add ADH into the solution of a), the concentration of ADH is 8.68 w / v%, stir at room temperature for 15 minutes;

[0115] c) Adjust the pH value of the solution of b) to 4.75 with hydrochloric acid, add anhydrous ethanol with a volume ratio of 1:1, stir at room temperature for 30 minutes;

[0116] d) Adjust the pH value of the solution of c) to 4.8 with sodium hydroxide, stir at room temperature for 2 hours;

[0117] e) Adjust the pH value of the solution of d) to 7;

[0118] f) Dialyze the solution of e) with a dialysis tube with MWCO = 3.5 kDa, first with 100 mM sodium chloride solution for 1 day, then with ethanol for 1 day, and finally with deionized water for 2 days;

[0119] g) Freeze dry the solution of f) to obtain HA-ADH, which is stored at 4°C;

[0120] h) Take HA-ADH, add it into deionized water and stir until completely dissolved to prepare a HA-ADH solution with a concentration of 0.5 w / v%;

[0121] i) Prepare PAMPS with a concentration of 5 wt%, adjust the pH value of the solution to 2.5, and stir at room temperature for 1 hour;

[0122] j) Mix the solution of h) and the solution of i), adjust the pH value of the mixed solution to 4.5, and stir at room temperature for 30 minutes;

[0123] k) EDC was added into the solution of j) with a concentration of 0.625 w / v%, the pH value of the solution was adjusted to 4.8, and the solution was stirred at room temperature for 5 minutes;

[0124] l) NHS was added into the solution of k) with a concentration of 0.5 w / v%, the pH value of the solution was adjusted to 5.5, and the solution was stirred at room temperature for 36 hours, and then the pH value of the solution was adjusted to 7;

[0125] m) The solution of l) was dialyzed with a dialysis tube with a MWCO of 3.5 kDa in 100 mM sodium chloride solution for 1 day, in ethanol for 1 day, and in deionized water for 2 days in sequence;

[0126] n) The solution of m) was lyophilized to obtain HA-PA, which was stored at 4°C.

[0127] The FTIR detection results of HA before modification, the prepared HA-ADH and HA-PA are shown in Figure 1 The H-NMR detection results of HA before modification and the prepared HA-PA are shown in Figure 2

[0128] Preparation of modified hydrogel precursor solution in Example 3

[0129] The HA-Tyr prepared in Example 1 was dissolved in deionized water to obtain a HA-Tyr solution with a concentration of 2 w / v%. The HA-PA prepared in Example 2 was dissolved in deionized water to obtain a HA-PA solution with a concentration of 0.1 w / v%. The HA-Tyr solution and the HA-PA solution were mixed in a ratio of 1:1 to obtain a HA-Tyr / HA-PA modified hydrogel precursor solution.

[0130] Preparation of HA-Tyr / HA-PA modified hyaluronic acid hydrogel in Example 4

[0131] The HA-Tyr / HA-PA modified hydrogel precursor solution prepared in Example 3 was added with 2.5 U / ml of horseradish peroxidase (HRP) and 600 μM of hydrogen peroxide, and vortexed.

[0132] The process of forming the gel is shown in Figure 3 From the experiment, it can be seen that the solution was converted into a hydrogel within 30 seconds after the addition of HRP and hydrogen peroxide to the HA-Tyr / HA-PA modified hydrogel precursor solution. After the gelation, the hydrogel at the bottom of the test tube did not spontaneously flow out.

[0133] The prepared HA-Tyr / HA-PA modified hyaluronic acid hydrogel was subjected to rheological analysis, and the results are shown in Figure 4 ​The storage modulus G' is greater than the loss modulus G", which proves that the HA-Tyr / HA-PA precursor aqueous solution is converted from solidification to gel state after the addition of HRP and H2O2, i.e., the hydrogel is successfully formed.

[0134] Comparative Example 1 Gelation performance test of HA-Tyr and hyaluronic acid-dopamine solution

[0135] The HA-Tyr prepared in Example 1 was taken and prepared into an HA-Tyr solution with a concentration of 2 w / v%. The hyaluronic acid modified with dopamine (HA-DA) was taken and prepared into an HA-DA solution with a concentration of 1 w / v% using deionized water.

[0136] The HA-Tyr and HA-DA solutions were mixed, and HRP and hydrogen peroxide were added. As shown in Figure 5 After the addition of HRP and hydrogen peroxide to the mixed solution of HA-Tyr and HA-DA, the solution failed to form a hydrogel and remained in a flowing solution state.

[0137] Although HA-Tyr contains groups that can form a gel, the gelation performance is lost after mixing HA-Tyr and HA-DA. The formulation of Comparative Example 1 is not suitable for the preparation of a hydrogel.

[0138] Example 5 Preparation of modified hyaluronic acid hydrogel loaded with exosomes

[0139] The HA-Tyr / HA-PA modified hydrogel precursor solution prepared in Example 3 was taken, and a certain amount of mesenchymal stem cell-derived exosomes was added. 2.5 U / ml of horseradish peroxidase (HRP) and 600 μM hydrogen peroxide were added to the solution c), and vortexed to form a hydrogel.

[0140] As can be seen, in the case of adding an appropriate amount of biologically active substances, the HA-Tyr / HA-PA modified hydrogel precursor solution can maintain its good gelation performance, and the formed hydrogel can uniformly and stably load the biologically active substances.

[0141] Example 6 Properties of HA-Tyr / HA-PA modified hyaluronic acid hydrogel

[0142] A common medical 2 mL syringe was used to take the HA-Tyr / HA-PA modified hyaluronic acid hydrogel prepared in Example 4, a 21G needle was installed, and the syringe was pushed. As shown in Figure 6 The HA-Tyr / HA-PA modified hyaluronic acid hydrogel can be sucked into the syringe and smoothly extruded through the 21G needle, which proves that the hydrogel has good injectability and is suitable for being applied to the joint cavity and other parts in the form of injection.

[0143] The tissue adhesion property of the HA-Tyr / HA-PA modified hyaluronic acid hydrogel was tested. As shown in Figure 7 , the hydrogel was attached to the finger joint, and it was observed that the hydrogel could adhere to the surface of the finger and bend with the bending of the finger joint, and would not detach from the surface of the finger. At the same time, the hydrogel could adhere to the surface of the metal medicine spoon and stretch to a certain extent without breaking, showing adhesion to metal materials. It is proved that the HA-Tyr / HA-PA modified hyaluronic acid hydrogel has good tissue adhesion and has the ability to remain in the injection site such as joint cavity.

[0144] Atomic force microscopy was performed on the HA-PA and HA-Tyr / HA-PA modified hyaluronic acid hydrogel. As shown in Figure 8 , HA-PA showed a brush-like structure with hyaluronic acid long chain as the backbone and PAMPS molecules as the side chain. After blending HA-PA with HA-Tyr, atomic force microscopy was performed, and the brush-like structure could still be observed. It is proved that the HA-Tyr / HA-PA modified hyaluronic acid hydrogel has a brush-like molecular structure similar to lubricin, which has the ability to reduce friction.

[0145] Example 7 Series of formulations of HA-Tyr / HA-PA modified hyaluronic acid hydrogel

[0146] The HA-Tyr prepared in Example 1 was prepared into a HA-Tyr solution with a concentration of 2 w / v%. The HA-PA prepared in Example 2 was prepared into a HA-PA solution with a concentration of 0.1 w / v%. According to the formulations shown in Table 1, the HA-Tyr, HA-PA solution and deionized water were mixed, and then HRP and hydrogen peroxide were added to initiate the formation of hydrogel.

[0147] Table 1 Series of formulations of modified hyaluronic acid hydrogel

[0148] Formulation No. 1 2 3 4 5 2% HA-Tyr 1ml 1ml 1ml 1ml 1ml 0.1% HA-PA 0.2ml 0.4ml 0.6ml 0.8ml 1ml 200 U / ml HRP 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml 3.75% H2O2 0.1ml 0.1ml 0.1ml 0.1ml 0.1ml H2O 0.8ml 0.6ml 0.4ml 0.2ml 1ml

[0149] The formation of hydrogel in Formulations 1-5 is shown in Figure 9 . The scanning electron microscopy (SEM) detection was performed on the hydrogel prepared in Formulations 1-5, and the results are shown in Figure 10 .

[0150] From the experimental results, it can be seen that within a certain range, the HA-Tyr solution and the HA-PA solution can be mixed in different proportions, and after adding HRP and hydrogen peroxide, hydrogel can be formed. Therefore, the hydrogel of the present disclosure has wide adaptability. According to the needs of actual application, the concentration can be flexibly adjusted within a certain range to prepare modified hyaluronic acid hydrogel with different hardness and mechanical strength.

[0151] The prepared HA-Tyr / HA-PA modified hyaluronic acid hydrogel presents a uniform reticular microstructure, and the cavity microstructure of the hydrogel is conducive to further loading of other bioactive substances.

[0152] As can be seen from the above examples, the present disclosure prepares two modified HAs (HA-Tyr and HA-PA), and further utilizes the two modified HAs to prepare an exosome-loaded modified hyaluronic acid hydrogel. In the preparation of the modified hyaluronic acid hydrogel, a traditional chemical crosslinking agent with cytotoxicity is not needed, but HRP is used to promote crosslinking to form a gel, so that the prepared hydrogel has good biocompatibility. The HA-PA has a brush-like molecular structure, which effectively improves the lubricity of the modified hyaluronic acid hydrogel.

[0153] The modified hyaluronic acid hydrogel of the present disclosure also has excellent injectability and tissue adhesion, so the modified hyaluronic acid hydrogel of the present disclosure can be safely and conveniently applied to joints and other parts, adheres to the applied parts to play a role in lubrication, buffering, etc.

[0154] Further, the modified hyaluronic acid hydrogel of the present disclosure can also load bioactive substances, such as mesenchymal stem cell-derived exosomes. Under the protection of the modified hyaluronic acid hydrogel, the half-life of the active substances is prolonged, and the biological effects can be stably and persistently exerted locally in the joints, thereby improving the treatment effect on osteoarthritis and other conditions.

[0155] Although the features of the present application have been shown and described in detail with reference to preferred embodiments, it will be understood by those skilled in the art that other changes can be made therein without departing from the spirit of the present application. The breadth and scope of the present disclosure should not be limited by any of the above exemplary embodiments.

Claims

1. A modified hyaluronic acid hydrogel formed from tyramine-modified hyaluronic acid and poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid under the action of horseradish peroxidase and hydrogen peroxide, the tyramine-modified hyaluronic acid being prepared by the following steps: dissolving hyaluronic acid in a buffer solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, stirring thoroughly, and then adding tyramine and stirring to react; after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain tyramine-modified hyaluronic acid; and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid being prepared by the following steps: dissolving hyaluronic acid in deionized water, adding adipic acid dihydrazide, adding anhydrous ethanol, and adjusting the pH to 7 as the reaction progresses; after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain adipic acid dihydrazide-modified hyaluronic acid; preparing an aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and an aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium, mixing the aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium, adjusting the pH of the mixed solution to 4-5, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, adjusting the pH to 4-6, stirring to react, and adjusting the pH of the solution after the reaction to 6.8-7.4; and after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid. wherein 2. A modified hyaluronic acid hydrogel loaded with a bioactive substance formed from tyramine-modified hyaluronic acid, poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid, and a bioactive substance under the action of horseradish peroxidase and hydrogen peroxide, the tyramine-modified hyaluronic acid being prepared by the following steps: dissolving hyaluronic acid in a buffer solution, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, stirring thoroughly, and then adding tyramine and stirring to react; after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain tyramine-modified hyaluronic acid; and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid being prepared by the following steps: dissolving hyaluronic acid in deionized water, adding adipic acid dihydrazide, adding anhydrous ethanol, and adjusting the pH to 7 as the reaction progresses; after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain adipic acid dihydrazide-modified hyaluronic acid; preparing an aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and an aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium, mixing the aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium, adjusting the pH of the mixed solution to 4-5, adding N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide, adjusting the pH to 4-6, stirring to react, and adjusting the pH of the solution after the reaction to 6.8-7.4; and after the reaction is completed, the reaction product is dialyzed and freeze-dried to obtain poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid. ​ ​ ​ ​ ​ ​ ​ ​ wherein, ​ ​ ​ ​ ​ ​ ​ After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid is prepared.

3. The bioactive substance-loaded modified hyaluronic acid hydrogel according to claim 2, wherein, The bioactive substance is one or more selected from mesenchymal stem cell-derived exosomes, curcumin, quercetin, and ganoderma polysaccharide.

4. A method for preparing a modified hyaluronic acid hydrogel, comprising the following steps: The tyramine-modified hyaluronic acid is prepared into an aqueous solution; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared into an aqueous solution; The aqueous solution of the tyramine-modified hyaluronic acid and the aqueous solution of the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid are mixed, horseradish peroxidase and hydrogen peroxide are added to the mixture, and vortexing is performed to obtain a modified hyaluronic acid hydrogel. The tyramine-modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in a buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and stirring is performed, and then tyramine is added, and stirring is performed for reaction; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the tyramine-modified hyaluronic acid is prepared; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in deionized water, adipic acid dihydrazide is added, anhydrous ethanol is added, and the pH is adjusted to 7 as the reaction progresses; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the adipic acid dihydrazide-modified hyaluronic acid is prepared; The aqueous solution of the adipic acid dihydrazide-modified hyaluronic acid and the aqueous solution of the poly-2-acrylamide-2-methylpropanesulfonic acid sodium are prepared, the aqueous solution of the adipic acid dihydrazide-modified hyaluronic acid and the aqueous solution of the poly-2-acrylamide-2-methylpropanesulfonic acid sodium are mixed, the pH of the mixture is adjusted to 4-5, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the pH is adjusted to 4-6, stirring is performed for reaction, and the pH of the solution after the reaction is adjusted to 6.8-7.4; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared.

5. A method for preparing a modified hyaluronic acid hydrogel loaded with a bioactive substance, comprising the following steps: The tyramine-modified hyaluronic acid is prepared into an aqueous solution; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared into an aqueous solution; The aqueous solution of the tyramine-modified hyaluronic acid, the aqueous solution of the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid, and the bioactive substance are mixed, horseradish peroxidase and hydrogen peroxide are added to the mixture, and vortexing is performed to obtain a modified hyaluronic acid hydrogel loaded with the bioactive substance, The tyramine-modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in a buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and stirring is performed, and then tyramine is added, and stirring is performed for reaction; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the tyramine-modified hyaluronic acid is prepared; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in deionized water, adipic acid dihydrazide is added, anhydrous ethanol is added, and the pH is adjusted to 7 as the reaction progresses; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the adipic acid dihydrazide modified hyaluronic acid is prepared; An aqueous solution of adipic acid dihydrazide modified hyaluronic acid and an aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are prepared, the aqueous solution of adipic acid dihydrazide modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are mixed, the pH of the mixed solution is adjusted to 4-5, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the pH is adjusted to 4-6, the reaction is stirred, and the pH of the solution after the reaction is adjusted to 6.8-7.4; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid is prepared.

6. A hydrogel precursor composition comprising an aqueous solution of tyramine modified hyaluronic acid and poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid, wherein The tyramine modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in a buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the solution is stirred thoroughly, and then tyramine is added, and the reaction is stirred; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the tyramine modified hyaluronic acid is prepared; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in deionized water, adipic acid dihydrazide is added, anhydrous ethanol is added, and the pH is adjusted to 7 as the reaction progresses; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the adipic acid dihydrazide modified hyaluronic acid is prepared; An aqueous solution of adipic acid dihydrazide modified hyaluronic acid and an aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are prepared, the aqueous solution of adipic acid dihydrazide modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are mixed, the pH of the mixed solution is adjusted to 4-5, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the pH is adjusted to 4-6, the reaction is stirred, and the pH of the solution after the reaction is adjusted to 6.8-7.4; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid is prepared.

7. A kit comprising an aqueous solution of tyramine modified hyaluronic acid, poly-2-acrylamide-2-methylpropanesulfonic acid sodium modified hyaluronic acid, horseradish peroxidase, hydrogen peroxide, and deionized water, wherein, The tyramine modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in a buffer, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the solution is stirred thoroughly, and then tyramine is added, and the reaction is stirred; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the tyramine-modified hyaluronic acid is prepared; The poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared by the following steps: The hyaluronic acid is dissolved in deionized water, adipic acid dihydrazide is added, anhydrous ethanol is added, and the pH is adjusted to 7 as the reaction progresses; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the adipic acid dihydrazide-modified hyaluronic acid is prepared; An aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and an aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are prepared, the aqueous solution of adipic acid dihydrazide-modified hyaluronic acid and the aqueous solution of poly-2-acrylamide-2-methylpropanesulfonic acid sodium are mixed, the pH of the mixture is adjusted to 4-5, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, the pH is adjusted to 4-6, the reaction is stirred, and the pH of the solution after the reaction is adjusted to 6.8-7.4; After the reaction is completed, the reaction product is dialyzed, freeze-dried, and the poly-2-acrylamide-2-methylpropanesulfonic acid sodium-modified hyaluronic acid is prepared.

8. Use of the modified hyaluronic acid hydrogel of claim 1 or the modified hyaluronic acid hydrogel loaded with a biologically active substance of claim 2 in the preparation of a preparation, a drug, or a medical device for treating osteoarthritis.

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