An active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration and its preparation method

Abstract

The present invention discloses a reactive oxygen species-responsive supramolecular hydrogel for promoting tissue regeneration and a preparation method thereof. First, a reactive oxygen species-responsive hyperbranched polymer with double bond end groups is synthesized; a hyperbranched polymer with cyclodextrin end groups is obtained through the reaction of the end group double bonds of the hyperbranched polymer with hexamethylenediamine-modified cyclodextrin; adamantane-modified hyaluronic acid is obtained through the transesterification reaction between adamantane acetic acid and hyaluronic acid; cyclodextrin-modified hyaluronic acid is obtained through the acylation reaction between hexamethylenediamine-modified cyclodextrin and hyaluronic acid; the prepared hyperbranched polymer, adamantane-modified hyaluronic acid, cyclodextrin-modified hyaluronic acid, and miRNA, drug or hyperbranched polylysine for promoting regeneration are dissolved in water, fully mixed, and incubated in situ to form a hydrogel. The hydrogel has good biocompatibility, can eliminate excessive reactive oxygen species in the microenvironment and promote regeneration; combining anti-inflammatory treatment strategies and pro-regeneration treatment strategies shows excellent therapeutic effects and has broad application prospects in tissue engineering regeneration.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical polymer materials, and particularly relates to a reactive oxygen species-responsive supramolecular hydrogel for promoting tissue regeneration and a preparation method thereof. Background Art

[0002] Reactive oxygen species are generated in various physiological activities of the human body and play an important role in regulating the redox balance. There are various forms of reactive oxygen species, mainly including superoxide anion radicals, hydrogen peroxide, hydroxyl radicals, and nitrogen oxide radicals, etc. There are various reactive oxygen species scavenging enzymes and small molecule antioxidants in the human body to maintain the dynamic balance of the generation and elimination of reactive oxygen species inside and outside cells. Once the balance is broken, especially when reactive oxygen species are overexpressed in the tissue microenvironment, chronic inflammation will be formed, ultimately leading to cell apoptosis and tissue damage, and inhibiting the normal tissue repair process. A large number of studies have shown that the mechanisms of various inflammatory diseases such as myocardial infarction, heart failure, spinal cord injury, cerebral infarction, arthritis, enteritis, etc. are closely related to the overexpressed reactive oxygen species in the tissue microenvironment. Therefore, it is of great significance to timely and effectively regulate the level of reactive oxygen species in the pathological microenvironment and maintain it at a normal level. In addition, according to the characteristics of overexpressed reactive oxygen species in the tissue microenvironment, designing a biomedical material containing a reactive oxygen species-responsive structure can not only be used as a drug delivery carrier for targeted treatment, but also can be used as a responsive material to inhibit inflammation and promote the normal repair of damaged tissues.

[0003] At present, some reactive oxygen species-responsive biomaterials, including hydrogels, patches, and nanoparticles, have certain positive effects on inflammation treatment. However, due to the limitations of anti-inflammatory treatment strategies and the biomaterials themselves, single anti-inflammatory regulation can prevent continuous damage, but cannot completely restore damaged tissues.

[0004] Various other treatment methods, such as regenerative therapies, including cell transplantation, RNA interference, and regenerative drugs, have been explored for the treatment of myocardial infarction and restoration of heart function, and have achieved varying degrees of success. MicroRNAs (miRNAs) are short single-stranded RNAs that can anneal with complementary sequences in mRNAs, thereby inhibiting protein expression. Single miRNAs participate in numerous mRNA targets and usually encode multiple components of complex intracellular networks. They are also involved in complex biological processes such as immune cell development and function, immune disorders, neural development, and neurological diseases. RNA interference-based targeted therapy is a very promising method for treating various diseases. Some other drugs involved in the regeneration pathway have also been developed and studied for the treatment of tissue diseases.

[0005] Anti-inflammatory treatment alone cannot fully restore damaged tissues, while promoting regeneration has great therapeutic prospects in tissue engineering. Therefore, we achieved long-term improvement of damaged tissues after disease by combining anti-inflammatory treatment using ROS-responsive biomaterials and regenerative treatment using pro-regenerative miRNAs and drugs. Summary of the invention

[0006] The purpose of the present invention is to provide an active oxygen responsive supramolecular hydrogel that promotes tissue regeneration and a preparation method thereof. The hydrogel can achieve active oxygen response and anti-inflammatory, releases pro-regeneration miRNA, drugs and hyperbranched polylysine (HBPL) by responsive degradation, and has good application prospects in promoting the treatment of inflammation-related diseases and tissue repair.

[0007] The invention discloses a method for preparing an active oxygen responsive supramolecular hydrogel for promoting tissue regeneration. The method comprises the following steps: obtaining an active oxygen responsive hyperbranched polymer containing a double bond end group through a Michael addition reaction between polyethylene glycol diacrylate (PEGDA) and an active oxygen responsive small molecule diamine; obtaining a hyperbranched polymer with a cyclodextrin end group through a Michael addition reaction between the end double bond of the hyperbranched polymer and hexamethylenediamine-modified cyclodextrin (CD-HDA); obtaining adamantane-modified hyaluronic acid through an ester exchange reaction between adamantane acetic acid and hyaluronic acid; obtaining cyclodextrin-modified hyaluronic acid through an acylation reaction between hexamethylenediamine-modified cyclodextrin and hyaluronic acid; dissolving the obtained hyperbranched polymer, adamantane-modified hyaluronic acid, cyclodextrin-modified hyaluronic acid and miRNA, drug or HBPL for promoting regeneration in water, fully mixing, and then forming a supramolecular active oxygen responsive hydrogel for promoting tissue regeneration through in situ incubation.

[0008] In the preparation method of the present invention, the active oxygen responsive small molecule can be a small molecule containing a diselenide bond or other structures having active oxygen responsiveness and having amino groups at both ends. Among them, the structural formula of the diamine small molecule containing a diselenide bond is:

[0009]

[0010] In the preparation method of the present invention, the specific preparation method of the hyperbranched polymer with cyclodextrin as the end group is as follows: Dissolve polyethylene glycol diacrylate and the active oxygen-responsive small molecule diamine in dimethyl sulfoxide, stir evenly, and react in an oil bath at 60-100 °C for 6-10 hours; Drop the reaction solution into the precipitant methyl tert-butyl ether for precipitation and discard the upper-layer waste liquid. Repeat this precipitation process 3-5 times. The obtained precipitated product is then rotary evaporated to remove a small amount of residual precipitant, and a hyperbranched polymer with double bonds at the end groups is obtained. Dissolve the obtained hyperbranched polymer and hexamethylenediamine-modified cyclodextrin in dimethyl sulfoxide, stir evenly, and react in an oil bath at 60-100 °C for 6-10 hours; Drop the reaction solution into the precipitant methyl tert-butyl ether for precipitation and discard the upper-layer waste liquid. Repeat this precipitation process 3-5 times. The obtained precipitated product is then rotary evaporated to remove a small amount of residual precipitant, dissolved in anhydrous methanol, and centrifuged repeatedly to remove the remaining hexamethylenediamine-modified cyclodextrin, and the methanol is removed by rotary evaporation to obtain a hyperbranched polymer with cyclodextrin as the end group.

[0011] In the preparation method of the present invention, the molar ratio of polyethylene glycol diacrylate to the active oxygen-responsive small molecule diamine is 2.2-2.5:1, the mass ratio of the active oxygen small molecule diamine to dimethyl sulfoxide is 1:16.5, and the molar ratio of the hyperbranched polymer with double bonds at the end groups to hexamethylenediamine-modified cyclodextrin is 1:2-2.5.

[0012] In the preparation method of the present invention, the specific preparation method of cyclodextrin-modified hyaluronic acid (CD-HA) is as follows: Add the strongly acidic ion exchange resin Dowex 50w×8-200 to the HA aqueous solution and stir at room temperature for 8 hours. Filter out the resin, and then adjust the pH to 7.02-7.05 with 0.2M aqueous tetrabutylammonium hydroxide (TBA-OH). The obtained aqueous solution is immediately frozen and lyophilized to obtain hyaluronic acid TBA salt (HA-TBA). Dissolve HA-TBA and hexamethylenediamine-modified cyclodextrin in anhydrous dimethyl sulfoxide. After complete dissolution, add a Carter condensing agent to the solution. After reacting for 3 hours, add deionized water to stop the reaction, transfer it to a dialysis bag, dialyze and purify, and freeze-dry to obtain.

[0013] In the specific preparation method of cyclodextrin-modified hyaluronic acid in the preparation method of the present invention, the mass-volume fraction of hyaluronic acid is 1-2%; the total mass of the strongly acidic ion exchange resin is at least 3 times that of hyaluronic acid; the molar amount of hexamethylenediamine-modified cyclodextrin is at least 1.3 times that of HA-TBA; the molar amount of the Carter condensing agent is the same as that of hexamethylenediamine-modified cyclodextrin. Dialysis is carried out at room temperature, and the water is changed once every 8-10 hours. After two weeks of dialysis in total, it is lyophilized.

[0014] In the preparation method of the present invention, the specific preparation method of adamantane-modified hyaluronic acid (Ad-HA) is as follows: strongly acidic ion exchange resin Dowex 50w×8-200 is added to the HA aqueous solution and stirred at room temperature for 8 hours. The resin is filtered off, and then the pH is adjusted to 7.02-7.05 with a 0.2M tetrabutylammonium hydroxide aqueous solution (TBA-OH). The obtained aqueous solution is immediately freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA). HA-TBA, 4-dimethylaminopyridine and adamantane acetic acid are dissolved in anhydrous dimethyl sulfoxide, and di-tert-butyl dicarbonate is added to the solution after complete dissolution. After reacting in an oil bath at 45°C for 20 hours, the mixture is transferred to a dialysis bag, dialyzed for purification, and freeze-dried to obtain.

[0015] In the preparation method of the present invention, in the specific preparation method of adamantane-modified hyaluronic acid, the mass volume fraction of hyaluronic acid is 1-2%; the total mass of the strong acid ion exchange resin is at least 3 times that of hyaluronic acid; the molar amount of adamantane acetic acid is at least 2.5 times that of HA-TBA; the molar amount of 4-dimethylaminopyridine is at least 0.8 times that of HA-TBA. The dialysis is carried out at room temperature, the water is changed every 8-10 hours, and the dialysis is carried out for two weeks.

[0016] In the preparation method of the present invention: further, a hyperbranched polymer with a final mass volume fraction of 5%-20%, Ad-HA with a final mass volume fraction of 5%-10%, CD-HA with a final mass volume fraction of 0-10%, miRNA with a drug or HBPL with a final mass volume fraction of 10-1000 μM is dissolved in ultrapure water, and the mixture is fully mixed and incubated for a period of time to obtain a supramolecular reactive oxygen responsive hydrogel. The drug may be 1,4-DPCA (HIF-1α pathway regulating drug), ganglioside, growth factor, etc.

[0017] The beneficial effects of the present invention are:

[0018] The present invention combines the anti-inflammatory treatment strategy with the tissue regeneration treatment strategy for the first time. The prepared hydrogel has good biocompatibility and shows excellent therapeutic effect. The prepared supramolecular active oxygen responsive hydrogel can effectively respond to degradation under the active oxygen environment. It can gel quickly, and the mechanical properties can be adjusted by adjusting the component content during the gelation process. The formed gel is injectable. Therefore, the gel has good application prospects in the treatment of diseases with excessive expression of active oxygen, such as myocardial infarction, heart failure, brain injury, spinal cord injury, cerebral infarction, arthritis, enteritis, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the H-NMR spectrum of a diamine small molecule containing a diselenide bond;

[0020] Figure 2 This is the GPC spectrum of the diselenide hyperbranched polymer HBPASe in Example 1;

[0021] Figure 3 1H NMR spectra of hyperbranched polymer HBPASe and CD-HBPASe containing diselenium in Example 1;

[0022] Figure 4 1H NMR spectra of cyclodextrin and adamantane-modified hyaluronic acid CD-HA and Ad-HA in Example 1;

[0023] Figure 5 Macrophotographs of the hydrogel prepared in a syringe and its injectable extrusion in Example 1;

[0024] Figure 6 Degradation of the hydrogel prepared in Example 1;

[0025] Figure 7 Regeneration promotion of the hydrogel prepared in Example 1. Detailed implementation manners

[0026] The technical solutions of the present invention are further described below in conjunction with embodiments, but these embodiments do not limit the present invention.

[0027] Example 1:

[0028] Synthesis of reactive oxygen species-responsive hyperbranched polymer CD-HBPASe: Weigh 2.5 g of polyethylene glycol diacrylate (PEGDA) and 0.5 g of selenocystamine in 7.5 mL of dimethyl sulfoxide, and stir well to dissolve; after reacting in an oil bath at 100 °C for 8 hours, cool the reaction solution to room temperature; drop the reaction solution into 5 times the volume of the precipitant methyl tert-butyl ether for precipitation, discard the upper waste liquid, and repeat this precipitation process 3-5 times. The precipitated product obtained is then subjected to rotary evaporation to remove a small amount of residual precipitant to obtain a hyperbranched polymer HBPASe with double bond end-capping. Dissolve 1 g of HBPASe and 2.5 g of CD-HDA in dimethyl sulfoxide, stir evenly, and react in an oil bath at 60-100 °C for 6-10 hours; drop the reaction solution into the precipitant methyl tert-butyl ether for precipitation and discard the upper waste liquid, repeat this precipitation process 3-5 times. The precipitated product obtained is then subjected to rotary evaporation to remove a small amount of residual precipitant. After adding methanol to dissolve the hyperbranched polymer, centrifuge three times to remove the excess CD-HDA, and then remove methanol by rotary evaporation to obtain the reactive oxygen species-responsive hyperbranched polymer CD-HBPASe.

[0029] Synthesis of cyclodextrin-modified hyaluronic acid CD-HA: Dissolve 3g of hyaluronic acid in 150ml of deionized water and stir evenly at room temperature; add 9.0g of Dowex 50WX8 ion exchange resin and stir at room temperature for 8 hours. Filter to remove the resin, and then adjust the pH to 7.02-7.05 with 0.2M tetrabutylammonium hydroxide aqueous solution (TBA-OH). The obtained aqueous solution is immediately freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA). Add 2.5g HA-TBA and 2.96g CD-HDA to a flask and dissolve in 125mL of anhydrous DMSO. After 1.06g of BOP is completely dissolved in 20mL of anhydrous DMSO, it is added to the HA-TBA / CD-HDA solution and stirred at room temperature for 3 hours. Cool with 10ml of cold deionized water, then transfer the solution to a dialysis bag with a molecular weight cutoff of 3500g / mol, change the water every 6-8 hours, dialyze for two weeks, and freeze-dry.

[0030] Synthesis of adamantane-modified hyaluronic acid Ad-HA: Dissolve 3g of hyaluronic acid in 150ml of deionized water and stir evenly at room temperature; add 9.0g of Dowex 50WX8 ion exchange resin and stir at room temperature for 8 hours. Filter to remove the resin, and then adjust the pH to 7.02-7.05 with 0.2M tetrabutylammonium hydroxide aqueous solution (TBA-OH). The obtained aqueous solution is immediately freeze-dried to obtain hyaluronic acid TBA salt (HA-TBA). Add 2.5g HA-TBA, 2.04g adamantane acetic acid and 0.32g 4-dimethylaminopyridine and dissolve in 125ml of anhydrous DMSO. After complete dissolution, add 0.35mL of di-tert-butyl carbonic anhydride to the reaction, stir in an oil bath at 45°C for 20 hours, and then transfer the solution to a dialysis bag with a molecular weight cutoff of 3500g / mol, change the water every 6-8 hours, dialyze for two weeks, and freeze-dry.

[0031] Preparation of reactive oxygen species responsive hydrogel: Dissolve 10% CD-HBPASe, 10% Ad-HA, 10% CD-HA and 200 μM mir19 in water and mix them thoroughly. Suck the mixture into a syringe and after mixing evenly, gel will form in the syringe.

[0032] The GPC spectrum and H NMR spectrum of HBPASe synthesized in this example are as follows: Figure 2 , Figure 3 The nuclear magnetic hydrogen spectra of HA-CD and HA-Ad synthesized in this example are shown in Figure 4 The macroscopic photograph of the hydrogel prepared in the syringe and its injectable extrusion in this example is shown in FIG. Figure 5As shown. The injectability of the hydrogel mainly makes the operation easier and more convenient. The reactive oxygen species-responsive hydrogel prepared in this example was placed in water and 50 mM H 2 O 2 aqueous solution respectively, and the degradation of the gel is as shown in Figure 6 As shown. The results show that the hydrogel degrades in both solutions, but degrades faster in 50 mM H 2 O 2 aqueous solution, indicating that the gel has both hydrolysis and reactive oxygen species degradation and can effectively respond to the reactive oxygen species environment. The therapeutic effect of the hydrogel is as shown in Figure 7 As shown. Compared with the simple anti-inflammatory hydrogel (R+ / M-) and the pro-regenerative hydrogel (R- / M+), the anti-inflammatory and pro-regenerative hydrogel (R+ / M+) prepared by the present invention has a better pro-regenerative effect on myocardial regeneration after myocardial infarction. Through immunofluorescence detection of mitotic markers phosphorylated histone 3 (pH3), Ki67 and Aurora B, it was found that the positive signals of pH3, Ki67 and Aurora B were significantly higher than those of other control groups, indicating that the combination of anti-inflammatory and pro-tissue regeneration treatment strategies has a more excellent pro-regenerative effect after myocardial infarction.

[0033] Example 2:

[0034] The synthesis of the reactive oxygen species-responsive hyperbranched polymer CD-HBPASe, cyclodextrin-modified hyaluronic acid CD-HA and adamantane-modified hyaluronic acid Ad-HA was carried out as in Example 1.

[0035] Preparation of the reactive oxygen species-responsive hydrogel: Dissolve CD-HBPASe with a final mass-volume fraction of 10%, Ad-HA with a final mass-volume fraction of 5%, CD-HA with a final mass-volume fraction of 5%, and 200 μM of mir19 in ultrapure water and mix well. Suck the mixed solution into a syringe, and after mixing evenly, a gel can be formed in the syringe.

[0036] Example 3:

[0037] The synthesis of the reactive oxygen species-responsive hyperbranched polymer CD-HBPASe, cyclodextrin-modified hyaluronic acid CD-HA and adamantane-modified hyaluronic acid Ad-HA was carried out as in Example 1.

[0038] Preparation of the reactive oxygen species-responsive hydrogel: Dissolve CD-HBPASe with a final mass-volume fraction of 5%, Ad-HA with a final mass-volume fraction of 5%, CD-HA with a final mass-volume fraction of 5%, and 200 μM of mir19 in ultrapure water and mix well. Suck the mixed solution into a syringe, and after mixing evenly, a gel can be formed in the syringe.

[0039] Example 4:

[0040] The synthesis of the reactive oxygen species-responsive hyperbranched polymer CD-HBPASe, cyclodextrin-modified hyaluronic acid CD-HA, and adamantane-modified hyaluronic acid Ad-HA was as described in Example 1.

[0041] Preparation of the reactive oxygen species-responsive hydrogel: CD-HBPASe with a final mass-volume fraction of 5%, Ad-HA with a final mass-volume fraction of 10%, CD-HA with a final mass-volume fraction of 10%, and 200 μg / mL of HBPL were dissolved in ultrapure water and thoroughly mixed. The mixture was aspirated into a syringe, and after thorough mixing, a gel formed inside the syringe.

Claims

1. Preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration, characterized in that, the preparation method is as follows: A hyperbranched polymer with active oxygen responsiveness containing double bond end groups is obtained through a Michael addition reaction between polyethylene glycol diacrylate and an active oxygen-responsive small molecule diamine; A hyperbranched polymer with cyclodextrin at the end group is obtained through a Michael addition reaction between the end group double bond of the hyperbranched polymer and hexamethylenediamine-modified cyclodextrin (CD-HDA); Adamantane-modified hyaluronic acid is obtained through a transesterification reaction between adamantane acetic acid and hyaluronic acid; Cyclodextrin-modified hyaluronic acid is obtained through an acylation reaction between hexamethylenediamine-modified cyclodextrin and hyaluronic acid; The prepared hyperbranched polymer, adamantane-modified hyaluronic acid, cyclodextrin-modified hyaluronic acid, and miRNA, drug or hyperbranched polylysine for promoting regeneration are dissolved in water, fully mixed, and a supramolecular active oxygen-responsive hydrogel for promoting tissue regeneration is formed through in-situ incubation.

2. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration according to claim 1, characterized in that, the preparation method of the hyperbranched polymer with cyclodextrin at the end group is as follows: Polyethylene glycol diacrylate and an active oxygen-responsive small molecule diamine are dissolved in dimethyl sulfoxide, stirred evenly, and subjected to an oil bath reaction at 60-100 °C for 6-10 hours; The reaction solution is dropped into the precipitant methyl tert-butyl ether for precipitation and the upper-layer waste liquid is discarded, and this precipitation process is repeated 3-5 times. The obtained precipitated product is then subjected to rotary evaporation to remove a small amount of residual precipitant, and a hyperbranched polymer with double bond end groups is obtained; The obtained hyperbranched polymer and hexamethylenediamine-modified cyclodextrin are dissolved in dimethyl sulfoxide, stirred evenly, and subjected to an oil bath reaction at 60-100 °C for 6-10 hours; The reaction solution is dropped into the precipitant methyl tert-butyl ether for precipitation and the upper-layer waste liquid is discarded, and this precipitation process is repeated 3-5 times. The obtained precipitated product is then subjected to rotary evaporation to remove the residual precipitant, dissolved in anhydrous methanol, and the remaining hexamethylenediamine-modified cyclodextrin is removed by repeated centrifugation, and methanol is removed by rotary evaporation to obtain a hyperbranched polymer with cyclodextrin at the end group.

3. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration according to claim 2, characterized in that, the molar ratio of the polyethylene glycol diacrylate to the active oxygen-responsive small molecule diamine is 2.2-2.5:1, the mass ratio of the active oxygen small molecule diamine to dimethyl sulfoxide is 1:16.5, and the molar ratio of the hyperbranched polymer with double bond end groups to hexamethylenediamine-modified cyclodextrin is 1:2-2.5; The active oxygen small molecule diamine is a small molecule with amino groups at both ends containing diselenide bonds, disulfide bonds, monoselenium, monosulfur or other structures with active oxygen responsiveness.

4. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration according to claim 1, characterized in that, The specific preparation method of cyclodextrin-modified hyaluronic acid (CD-HA) is as follows: Add strongly acidic ion exchange resin Dowex 50w×8-200 to the HA aqueous solution, stir at room temperature for 8 hours, filter out the resin, and then adjust the pH to 7.02-7.05 with 0.2M aqueous solution of tetrabutylammonium hydroxide (TBA-OH). The obtained aqueous solution is immediately frozen and lyophilized to obtain hyaluronic acid TBA salt (HA-TBA). Dissolve HA-TBA and hexamethylenediamine cyclodextrin in anhydrous dimethyl sulfoxide. After complete dissolution, add Carter condensing agent to the solution. After reacting for 3 hours, add deionized water to stop the reaction, transfer it to a dialysis bag, dialyze and purify, and then freeze-dry to obtain it.

5. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration as described in claim 4, characterized in that, in the specific preparation method of the cyclodextrin-modified hyaluronic acid (CD-HA): the mass-volume fraction of hyaluronic acid is 1-2%; the total mass of the strongly acidic ion exchange resin is at least 3 times that of hyaluronic acid; the molar amount of hexamethylenediamine cyclodextrin is at least 1.3 times that of HA-TBA; the molar amount of Carter condensing agent is the same as that of hexamethylenediamine cyclodextrin; dialysis is carried out at room temperature, and the water is changed every 8-10 hours. After 14 days of dialysis, it is freeze-dried.

6. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration as described in claim 1, characterized in that, The specific preparation method of adamantane-modified hyaluronic acid (Ad-HA) is as follows: Add strongly acidic ion exchange resin Dowex50w×8-200 to the HA aqueous solution, stir at room temperature for 8 hours, filter out the resin, and then adjust the pH to 7.02-7.05 with 0.2M aqueous solution of tetrabutylammonium hydroxide (TBA-OH). The obtained aqueous solution is immediately frozen and lyophilized to obtain hyaluronic acid TBA salt (HA-TBA). Dissolve HA-TBA, 4-dimethylaminopyridine and adamantane acetic acid in anhydrous dimethyl sulfoxide. After complete dissolution, add di-tert-butyl dicarbonate to the solution. After reacting in an oil bath at 45°C for 20 hours, transfer it to a dialysis bag, dialyze and purify, and then freeze-dry to obtain it.

7. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration as described in claim 6, characterized in that, in the specific preparation method of the adamantane-modified hyaluronic acid (Ad-HA): the mass-volume fraction of hyaluronic acid is 1-2%; the total mass of the strongly acidic ion exchange resin is at least 3 times that of hyaluronic acid; the mass of adamantane acetic acid is 0.5-2.5 times that of HA-TBA; the molar amount of 4-dimethylaminopyridine is at least 0.5-2.5 times that of HA-TBA; dialysis is carried out at room temperature, and the water is changed every 8-10 hours. After two weeks of dialysis, it is freeze-dried.

8. The preparation method of an active oxygen-responsive supramolecular hydrogel for promoting tissue regeneration as described in claim 1, characterized in that, The miRNA is at least one of mir199a-3p, mir-590-3p, and mir19a / b, and the drug is at least one of prolyl-4-hydroxylase inhibitors, cerebrolysin, and brain protein hydrolysate.

9. The preparation method of a reactive oxygen species-responsive supramolecular hydrogel for promoting tissue regeneration according to claim 1, characterized in that: dissolve hyperbranched polymer with a final mass volume fraction of 1%-20%, Ad-HA (adamantane-modified hyaluronic acid) with a mass volume fraction of 5%-10%, CD-HA (cyclodextrin-modified hyaluronic acid) with a mass volume fraction of 5%-10%, 10-1000 μmol of miRNA, drug or hyperbranched polylysine in ultrapure water, fully mix and incubate for a period of time to obtain a reactive oxygen species-responsive hydrogel for promoting regeneration.

10. The use of the hydrogel prepared by the method according to claim 1, characterized in that it is used for preparing drugs for treating diseases with overexpression of reactive oxygen species in tissue microenvironment, and the diseases include myocardial infarction, heart failure, brain injury, spinal cord injury, cerebral infarction, arthritis, and enteritis.

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