Long-acting high-dynamic hydrazone bond hydrogel as well as preparation method and application thereof

Abstract

The invention discloses long-acting high-dynamic hydrazone bond hydrogel as well as a preparation method and application thereof, and particularly relates to the technical field of biomedical materials. The preparation method comprises the following steps: taking 1-adamantane acetic acid and adipic dihydrazide modified sodium hyaluronate and benzaldehyde-terminated eight-arm polyethylene glycol as carrier raw materials; the hydrazone bond hydrogel with relatively high stability is prepared through a coupling reaction of an aldehyde group of benzaldehyde-terminated eight-arm polyethylene glycol and a hydrazide group of 1-adamantane acetic acid and adipic dihydrazide modified sodium hyaluronate. The beta-cyclodextrin modified m-phenylenediamine catalyst is dynamically combined into the carrier through reversible host-guest complexation between beta-cyclodextrin and 1-adamantane acetic acid, so that the long-acting high-dynamic hydrazone bond hydrogel is obtained, and the requirement of three-dimensional culture of stem cells on mechanical properties can be met; and network dynamics can be maintained for a long time, so that stem cell spreading is supported, mechanical signal transduction is activated, osteogenesis-related gene expression is promoted, and stem cell osteogenesis differentiation capability is enhanced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a long-acting high-dynamic hydrazone bond hydrogel and a preparation method and application thereof. BACKGROUND

[0002] Cell therapy based on human mesenchymal stem cells (hMSCs) shows great therapeutic potential in the repair and regeneration of bone and other tissues. However, direct injection of hMSCs into the injury site often results in low cell survival rate, poor implantation efficiency and limited directional differentiation ability, which seriously affects its clinical efficacy. Hydrogel, as a kind of biomaterial with controllable physical and biological properties and good biocompatibility, can deliver therapeutic cells or growth factors to bone defect sites and is considered as a promising scaffold material for bone tissue engineering. However, the hydrogels currently used as stem cell scaffolds are mostly composed of "static" networks cross-linked by permanent covalent bonds, which lack dynamic adaptability, limiting the remodeling ability of stem cells to the matrix and their related functions in the process of tissue development.

[0003] Since dynamic hydrazone bond combines the stability of covalent bond and the reversibility of non-covalent bond, its introduction into the gel network can construct a hydrogel with both macroscopic stability and microscopic dynamics. However, the formation and dissociation kinetics of hydrazone bond is slow, which makes the microscopic dynamic performance of gel network insufficient, and it is difficult to quickly respond to the physiological activities of the loaded stem cells. By introducing organic amine catalyst into the hydrazone bond hydrogel system, the dynamic exchange reaction of hydrazone bond can be effectively accelerated, and the structural dynamics of hydrogel can be significantly improved. However, a new problem arises, the free catalyst is easy to diffuse and lose from the hydrogel network during cell culture, which leads to rapid decay of the dynamic performance of hydrogel, seriously restricting the actual application effect of hydrazone bond hydrogel as a stem cell carrier. In summary, if the long-term retention of organic amine catalyst in the hydrazone bond hydrogel network can be achieved without compromising its activity, it is expected to develop a long-acting high-dynamic hydrazone bond hydrogel to meet the urgent needs of stem cell therapy for functional hydrogel carriers.

[0004] Chinese patent application CN108578707A discloses a kind of supramolecular hydrogel loaded with indomethacin and its preparation method, the hydrogel is prepared by chemical modification to β-cyclodextrin, a new cyclodextrin derivative is prepared;Then indomethacin is condensed with amido modified cyclodextrin derivative, and prodrug molecule indo-2N-β-CD is synthesized;Finally, with α-cyclodextrin, Pluronic F127, indo-2N-β-CD as building block, in aqueous solution, first, the interaction between the cavity of cyclodextrin and the molecular chain of F127 is used to prepare quasi-polyrotaxane loaded with hydrophobic drug, then the hydrogen bond between the hydroxyl groups in the cyclodextrin molecule is used to further expand into a three-dimensional new supramolecular hydrogel material, but the patent does not improve the dynamicity of the hydrogel structure, so it still needs to be improved. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a long-acting high-dynamic hydrazone bond hydrogel and its preparation method and application, which effectively solves the technical problem of dynamicity attenuation caused by catalyst loss in the cell culture process of the existing hydrazone bond hydrogel system.

[0006] The present application solves the above technical problems by the following technical means: The present application provides a preparation method of a long-acting high-dynamic hydrazone bond hydrogel, comprising the following steps: Mixing the aqueous solution of sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide with β-cyclodextrin modified m-phenylenediamine to obtain a reaction solution, then adding a benzaldehyde-capped eight-arm polyethylene glycol aqueous solution, and incubating to obtain the long-acting high-dynamic hydrazone bond hydrogel. The structural formula of the β-cyclodextrin modified m-phenylenediamine is: .

[0007] Preferably, the preparation method of the β-cyclodextrin modified m-phenylenediamine is as follows: First, β-cyclodextrin (βCD) is reacted with p-toluenesulfonyl chloride, and then reacted with 1,3-propanediamine to obtain mono-(6-propanediamino-6-deoxy)-βCD;Through amide condensation reaction, 3,5-diaminobenzoic acid protected by tert-butyloxycarbonyl is bonded to mono-(6-propanediamino-6-deoxy)-βCD;Using trifluoroacetic acid to remove the tert-butyloxycarbonyl protecting group, the long-acting high-dynamic hydrazone bond hydrogel is obtained.

[0008] Preferably, the preparation method of the sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide is as follows: Sodium hyaluronate is treated into hyaluronic acid by using 732 resin, and then hyaluronic acid tetrabutylammonium salt is obtained after neutralization by tetrabutylammonium hydroxide;Then, 1-adamantane acetic acid is grafted by esterification reaction to obtain 1-adamantane acetic acid modified sodium hyaluronate;Then, adipic acid dihydrazide is grafted by amide condensation reaction, and the long-acting high-dynamic hydrazone bond hydrogel is obtained.

[0009] Preferably, the preparation method of the benzaldehyde-terminated eight-arm polyethylene glycol is as follows: The 4-formyl benzoic acid is modified to the end of the eight-arm polyethylene glycol by esterification reaction using N, N'-dicyclohexyl carbodiimide and 4-dimethylamino pyridine as catalyst, and the product is purified by reprecipitation and dialysis method.

[0010] Preferably, the molar ratio of the β-cyclodextrin modified m-phenylenediamine to 1-adamantane acetic acid is 1:0.8~1:2, and the molar ratio of the aldehyde group of the benzaldehyde-terminated eight-arm polyethylene glycol to the hydrazine group of adipic acid dihydrazide is 1:1~1:2. Further preferably, the molar ratio of the β-cyclodextrin modified m-phenylenediamine to 1-adamantane acetic acid is 1:1, and the molar ratio of the aldehyde group of the benzaldehyde-terminated eight-arm polyethylene glycol to the hydrazine group of adipic acid dihydrazide is 1:1.

[0011] Beneficial effect: when the β-cyclodextrin modified m-phenylenediamine and 1-adamantane acetic acid are included at a molar ratio of 1:1, and the aldehyde group of the benzaldehyde-terminated eight-arm polyethylene glycol reacts with the hydrazine group of adipic acid dihydrazide at a molar ratio of 1:1, the ratio will make the hydrogel produce the best crosslinking state and network dynamics, providing a suitable microenvironment for stem cell growth.

[0012] Preferably, the sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide has a weight average molecular weight of 58 kDa~1729 kDa. Preferably, the substitution degree of the sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide is DS -Ad =0.10~0.25, and DS -ADH =0.15~0.35.

[0013] Preferably, the benzaldehyde-terminated eight-arm polyethylene glycol has a weight average molecular weight of 11 kDa~41 kDa. Preferably, the substitution degree DS -CHO of the benzaldehyde-terminated eight-arm polyethylene glycol is 5~7.92, calculated based on the number of aldehyde groups on each benzaldehyde-terminated eight-arm polyethylene glycol molecule.

[0014] Preferably, the concentration of the β-cyclodextrin modified m-phenylenediamine in the reaction solution is 3.18~10.39 mM.

[0015] Beneficial effect: In order to ensure the support effect of the hydrazone bond hydrogel on the three-dimensional culture of stem cells, the concentration of the beta-cyclodextrin modified m-phenylenediamine catalyst in the long-acting high-dynamic hydrazone bond hydrogel is 3.18-10.39 mM. If the amount of catalyst is less than 3.18 mM, it is difficult to effectively promote the reversible dissociation and reformation process of the hydrazone bond, resulting in a slow dynamic response rate of the gel network; and if the amount of catalyst is higher than 10.39 mM, the excess catalyst cannot be effectively anchored by the gel network and is quickly released, and may cause potential side effects.

[0016] Preferably, the solid content of the long-acting high-dynamic hydrazone bond hydrogel is 4.5 w / v%-8.4 w / v%.

[0017] Beneficial effect: In order to obtain a hydrogel with better state, the solid content of the long-acting high-dynamic hydrazone bond hydrogel is 4.5 w / v%-8.4 w / v%. When the solid content is less than 4.5 w / v%, it cannot provide sufficient mechanical support, and when the solid content is greater than 8.4 w / v%, it will make the hydrogel too hard, hindering the spreading and proliferation of stem cells. Preferably, the incubation temperature is 20-37℃, and the incubation time is 10-20 min.

[0018] The application also provides a long-acting high-dynamic hydrazone bond hydrogel prepared by the above method.

[0019] The application also provides the application of the long-acting high-dynamic hydrazone bond hydrogel prepared by the above method in the three-dimensional culture of stem cells.

[0020] The beneficial effects of the application are: 1. The application provides a long-acting high-dynamic hydrazone bond hydrogel, a preparation method thereof and an application thereof. The long-acting high-dynamic hydrazone bond hydrogel is prepared by using 1-adamantane acetic acid and adipic acid dihydrazide modified sodium hyaluronate (HA-Ad-ADH) and benzaldehyde capped eight-arm polyethylene glycol (PEG8-BA) as carrier raw materials, and through a hydrazone bond coupling reaction of the aldehyde group of PEG8-BA and the hydrazine group of HA-Ad-ADH, a hydrazone bond hydrogel with high stability is prepared, and through a reversible host-guest complexation between the beta-cyclodextrin cavity of the beta-cyclodextrin modified m-phenylenediamine (betaCD-mPD) catalyst and 1-adamantane acetic acid, the betaCD-mPD catalyst is dynamically combined into the carrier, thereby obtaining the long-acting high-dynamic hydrazone bond hydrogel.

[0021] 2. The long-acting high-dynamic hydrazone bond hydrogel prepared by the application has more excellent comprehensive performance than traditional hydrogels: on the one hand, the high hydrolysis resistance of the hydrazone bond ensures the structural and mechanical stability of the hydrogel, meeting the requirement of three-dimensional culture of stem cells on mechanical properties; on the other hand, the problem of insufficient micro-dynamic performance of traditional hydrazone bond hydrogels is solved by means of the'reversible anchoring catalyst' strategy, so that the hydrogel can maintain network dynamics for a long time, thereby supporting the spreading of stem cells, activating mechanical signal transduction, promoting the expression of osteogenesis-related genes, and enhancing the osteogenic differentiation ability of stem cells.

[0022] 3. The application constructs a hydrazone bond hydrogel system with long-term structural stability and high dynamics, which can effectively promote the adhesion, spreading and osteogenic differentiation process of stem cells.

[0023] Of course, the implementation of any product or method of the application does not necessarily require all the advantages described above. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 It is the nuclear magnetic hydrogen spectrum of betaCD-mPD in the application, and the deuterated solvent is D2O; Figure 2 It is the nuclear magnetic hydrogen spectrum of HA-Ad and HA-Ad-ADH in the application, and the deuterated solvent is D2O, wherein a is the nuclear magnetic hydrogen spectrum of HA-Ad, and b is the nuclear magnetic hydrogen spectrum of HA-Ad-ADH; Figure 3 It is the nuclear magnetic hydrogen spectrum of PEG8-BA in the application, and the deuterated solvent is CDCl3; Figure 4 It is a schematic diagram of the reversible anchoring catalyst accelerating the exchange kinetics of the hydrazone bond in the long-acting high-dynamic hydrazone bond hydrogel in Example 5 of the application; Figure 5 It is a preparation flowchart of the long-acting high-dynamic hydrazone bond hydrogel in Example 5 of the application; Figure 6 It is the standard curve of betaCD-mPD in Example 1 of the application; Figure 7 It is the standard curve of DABA in Comparative Example 2 of the application; Figure 8 It is a photo of the preparation of the long-acting high-dynamic hydrazone bond hydrogel from the component solutions in Example 5 of the application; Figure 9 It is a rheometer time scan graph of the hydrazone bond hydrogel prepared in Example 3, 4, 5, 6 and Comparative Example 1, 2 of the application; Figure 10 It is a rheometer frequency scan graph of the hydrazone bond hydrogel prepared in Example 3, 4, 5, 6 and Comparative Example 1, 2 of the application; Figure 11Rheometer stress relaxation mode scanning diagram of hydrazone bond hydrogel prepared in Example 3, 4, 5, 6 and Comparative Example 1, 2 of the present application; Figure 12 Self-repairing test diagram of hydrazone bond hydrogel prepared in Example 5 and Comparative Example 1 of the present application; Figure 13 Self-adapting test diagram of hydrazone bond hydrogel prepared in Example 5 and Comparative Example 1 of the present application; Figure 14 Mass degradation curve of hydrazone bond hydrogel prepared in Example 5 and Comparative Example 1 of the present application; Figure 15 Release curve of catalyst in hydrazone bond hydrogel prepared in Example 5 and Comparative Example 2 of the present application; Figure 16 Rheometer frequency scanning diagram and stress relaxation mode scanning diagram of hydrazone bond hydrogel prepared in Example 5 and Comparative Example 2 of the present application after PBS extraction; Figure 17 Blood compatibility evaluation results of DABA, βCD-mPD and long-acting high-dynamic hydrazone bond hydrogel in Example 5 of the present application; Figure 18 Hemolysis rate determination results of DABA, βCD-mPD and long-acting high-dynamic hydrazone bond hydrogel in Example 5 of the present application; Figure 19 Cytotoxicity determination results of DABA, βCD-mPD and long-acting high-dynamic hydrazone bond hydrogel in Example 5 of the present application; Figure 20 F-actin immunofluorescence staining image of hMSCs in hydrazone bond hydrogel prepared in Example 5 and Comparative Example 2 of the present application; Figure 21 YAP immunofluorescence staining image of hMSCs in hydrazone bond hydrogel prepared in Example 5 and Comparative Example 2 of the present application; Figure 22 Evaluation of osteogenic differentiation ability of hMSCs in hydrazone bond hydrogel prepared in Example 5 and Comparative Example 2 of the present application. DETAILED DESCRIPTION

[0025] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application. Unless otherwise defined, the professional terms used below are consistent with the meanings understood by those skilled in the art.

[0026] The test materials and reagents used in the following examples, unless otherwise specified, can be obtained commercially or prepared by known methods.

[0027] Unless otherwise specified, the quantitative tests in the following examples are set up with more than three repeated experiments, and the results are averaged.

[0028] For the problems mentioned in the background of the present application: when human mesenchymal stem cells (hMSCs) are directly injected into the injury site, there are problems of low cell survival rate, poor implantation efficiency and limited directional differentiation ability. Although hydrogel is an ideal bone tissue engineering scaffold material, the existing stem cell scaffold hydrogel is mainly composed of a "static" network cross-linked by permanent covalent bonds, which lacks dynamic adaptability and limits the remodeling of stem cell matrix and the function. Although the hydrazone bond hydrogel has macroscopic stability and microscopic dynamics, the slow exchange kinetics of the hydrazone bond leads to insufficient dynamic performance, and the introduction of free organic amine catalyst can improve the dynamic performance, but the free diffusion and loss of the catalyst can quickly reduce the dynamic performance, which seriously restricts the actual application of the hydrogel as a stem cell carrier. Therefore, the present application provides a long-acting high-dynamic hydrazone bond hydrogel and a preparation method and application thereof.

[0029] The present application first provides a preparation method of a long-acting high-dynamic hydrazone bond hydrogel, comprising the following steps: The β-cyclodextrin (βCD) is reacted with p-toluenesulfonyl chloride to obtain mono-6-O-(p-toluenesulfonyl)-βCD, which is then reacted with 1,3-propanediamine to obtain mono-(6-propanediamino-6-deoxy)-βCD (βCD-NH2). Then, the tert-butoxycarbonyl (Boc) protected 3,5-diaminobenzoic acid is linked to βCD-NH2 through an amide condensation reaction. Finally, the Boc protecting group is removed using trifluoroacetic acid to obtain a β-cyclodextrin modified m-phenylenediamine (βCD-mPD) catalyst.

[0030] The hyaluronic acid sodium (HA-Ad-ADH) aqueous solution modified by 1-adamantane acetic acid and adipic acid dihydrazide is mixed with the βCD-mPD, the β-cyclodextrin and the 1-adamantane acetic acid produce a host-guest complexation, forming a host-guest assembly solution containing the catalyst. Then, the benzaldehyde-terminated eight-arm polyethylene glycol (PEG8-BA) aqueous solution is added to the above mixture, and the mixture is incubated at 20-37℃ for 10-20 min. The aldehyde group of the PEG8-BA and the hydrazine group of the HA-Ad-ADH undergo a hydrazone bond coupling reaction to obtain a hydrogel carrier, and the catalyst is reversibly anchored to the carrier to obtain a long-acting high-dynamic hydrazone bond hydrogel.

[0031] The long-acting high-dynamic hydrazone bond hydrogel prepared by the technical scheme has the advantages of dynamicity and stability. The high hydrolysis resistance of the hydrazone bond guarantees the structural and mechanical stability of the hydrogel, and meets the requirement of the three-dimensional culture of stem cells on mechanical properties. The "reversible anchor catalyst" strategy ensures the persistence of dynamic performance in the cell culture period, thereby supporting the spreading of stem cells, activating mechanical signal transduction, promoting osteogenic gene expression, and providing a high-quality carrier for constructing an ideal three-dimensional culture platform suitable for stem cell growth.

[0032] The technical effects of the present application are described below through specific examples and comparative examples.

[0033] The present application first synthesizes β-cyclodextrin modified m-phenylenediamine (βCD-mPD), hyaluronic acid sodium modified by 1-adamantane acetic acid and adipic dihydrazide (HA-Ad-ADH), and benzaldehyde-capped eight-arm polyethylene glycol (PEG8-BA), as follows: 1. Synthesis of βCD-mPD: β-cyclodextrin (60 g, 52.86 mmol) was dissolved in 600 mL of 0.4 M sodium hydroxide solution, and p-toluenesulfonyl chloride (42.33 g, 222.03 mmol) was added in batches under ice water bath within 10 min, and then the reaction was continued under ice water bath for 45 min. After the reaction was completed, the reaction solution was collected by filtration, the pH was adjusted to 7 with concentrated hydrochloric acid solution, and then the stirring was continued at room temperature for 1 h. The white precipitate was collected by filtration and washed with cold water (120 mL x 2) and acetone (240 mL x 1). The precipitate was collected by centrifugation, and after vacuum drying at 35℃, the crude product was subjected to three recrystallization operations. First, the crude product was dissolved in 19.2 mL of dimethyl sulfoxide under heating, and after cooling to room temperature, it was poured into 240 mL of cold water for reprecipitation; second, the collected precipitate was mixed with 96 mL of pure water, heated to dissolve, and after cooling to room temperature, it was transferred to a 4℃ refrigerator for recrystallization, the solid was collected by filtration, and washed with cold water (72 mL x 2) and acetone (144 mL x 1), and dried at 35℃ under vacuum to obtain single-6-O- (p-toluenesulfonyl)-βCD (βCD-OTs).

[0034] βCD-OTs (4 g, 3.07 mmol) was dissolved in 24 mL of 1,3-propanediamine solution, and stirred at 80℃ for 18 h. After most of the 1,3-propanediamine was removed by rotary evaporation, it was dissolved in 20 mL of 25 v / v% methanol aqueous solution and poured into 200 mL of cold acetone for reprecipitation. The precipitate was collected by filtration, dissolved in 5 mL of deionized water, and poured into 100 mL of cold acetone for reprecipitation again. This was repeated three times. The precipitate was collected and dried at 35℃ under vacuum to obtain single- (6-propanediamine-6-deoxy)-βCD (βCD-NH2).

[0035] βCD-NH2(500 mg, 0.42 mmol) and tert-butoxycarbonyl-protected 3,5-diaminobenzoic acid (192.94 mg, 0.55 mmol) were dissolved in 25 mL of N,N-dimethylformamide, and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (284.91 mg, 0.55 mmol) and anhydrous triethylamine (76 μL, 0.55 mmol) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the reaction was precipitated into 125 mL of diethyl ether, and the precipitate was collected by centrifugation, and then the precipitate was dissolved in 3 mL of N,N-dimethylformamide and precipitated again into diethyl ether, and this was repeated three times. The collected precipitate was washed with diethyl ether (50 mL x 2) and dried under vacuum at 35°C to obtain 5-bis((tert-butoxycarbonyl)amino)benzoylated βCD-NH2(βCD-BABA-Boc).

[0036] Subsequently, 500 mg of βCD-BABA-Boc was dissolved in 3 mL of trifluoroacetic acid, and the reaction was stirred at room temperature for 1 h. After the reaction was completed, the reaction was precipitated into 15 mL of diethyl ether, and the precipitate was collected by centrifugation and washed with diethyl ether (120 mL x 2). The precipitate was collected by centrifugation, and 3 mL of water and ammonia were sequentially added thereto, and the reaction was stirred at room temperature for 0.5 h to remove salts. After the reaction was completed, the reaction was precipitated into 25 mL of ethanol, and the precipitate was collected by centrifugation, dissolved in 3 mL of water, and precipitated again into ethanol, and this was repeated three times. After drying under vacuum at 35°C, the final product βCD-mPD was obtained. The structural formula thereof is: Figure 1

[0037] ​​2. Synthesis of HA-Ad-ADH: Sodium hyaluronate (500 mg, 1.24 mmol of repeating units) was dissolved in 50 mL of deionized water, and 2.5 g of 732 resin was added. The mixture was stirred at room temperature for 4 h, and the supernatant was collected by centrifugation. The pH was adjusted to 7 with 25% tetrabutylammonium hydroxide aqueous solution, and sodium hyaluronate tetrabutylammonium salt was obtained by freeze-drying. Sodium hyaluronate tetrabutylammonium salt (500 mg, 0.78 mmol of repeating units) and 1-adamantaneacetic acid (453.33 mg, 2.33 mmol) were dissolved in 78 mL of dimethyl sulfoxide, and 4-dimethylaminopyridine (71.27 mg, 0.58 mmol) and di-tert-butyl dicarbonate (268 μL, 1.17 mmol) were added. The mixture was stirred at 45°C for 24 h in the dark, and the reaction was stopped by dialysis against dimethyl sulfoxide, 1M NaCl solution, and deionized water in sequence using dialysis bags with a molecular weight cut-off of 8000-14000 Da. The final product, 1-adamantaneacetic acid-modified sodium hyaluronate (HA-Ad), was obtained by freeze-drying. The structural formula is shown in: The 1H NMR spectrum (D2O as the deuterated solvent) is shown in FIG. a of Figure 2 The degree of substitution of 1-adamantaneacetic acid groups on sodium hyaluronate, DS -Ad was 0.17, as confirmed by 1H NMR.

[0038] A mixed solvent was prepared by mixing 4-morpholine ethanesulfonic acid aqueous solution (MES·H2O, 10 mM) and dimethyl sulfoxide (DMSO) at a volume ratio of 1:2. HA-Ad (500 mg, 1.1 mmol of repeating units) and adipic acid dihydrazide (960.73 mg, 5.52 mmol) were dissolved in 100 mL and 200 mL of the mixed solvent, respectively, to obtain solution A and solution B. Solution A was added to solution B at a constant rate, followed by the addition of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (74.01 mg, 0.39 mmol) and N-hydroxysuccinimide (44.43 mg, 0.39 mmol). The mixture was stirred at room temperature in the dark for 24 h, dialyzed against deionized water using dialysis bags with a molecular weight cut-off of 8000-14000 Da, and freeze-dried to obtain the final product, HA-Ad-ADH. The structural formula is shown in: The 1H NMR spectrum (D2O as the deuterated solvent) is shown in FIG. b of Figure 2 The degree of substitution of adipic acid dihydrazide groups on sodium hyaluronate, DS -ADH was 0.21, as confirmed by 1H NMR.

[0039] 3. Synthesis of PEG8-BA: Eight-arm polyethylene glycol (M W=10 kDa, 500 mg, 0.4 mmol -OH) and N, N'-dicyclohexyl carbodiimide (412.66 mg, 2 mmol) were dissolved in 10 mL of tetrahydrofuran to obtain solution C. And 4-formylbenzoic acid (240.21 mg, 1.6 mmol) and 4-dimethylamino pyridine (12.22 mg, 0.1 mmol) were dissolved in 15 mL of tetrahydrofuran to obtain solution D, and solution C was added to solution D at a constant speed in an ice bath for 30 min, and then the reaction was stirred at room temperature for 24 h. The reaction solution was collected by filtration, and the reaction solution was added dropwise into 75 mL of cold ether for reprecipitation. The precipitate was collected by centrifugation, dissolved in 5 mL of tetrahydrofuran, and then added dropwise into 50 mL of cold ether for reprecipitation. The precipitate was collected by centrifugation and dissolved in 2 mL of tetrahydrofuran. The final product PEG8-BA was obtained by dialysis against deionized water using a dialysis bag with a molecular weight cutoff of 3500 Da and freeze-drying. The structural formula of PEG8-BA is as follows: The nuclear magnetic resonance spectrum (deuterated solvent CDCl3) is as shown in Figure 3 The degree of substitution of aldehyde groups in the eight-arm polyethylene glycol was confirmed by nuclear magnetic resonance to be DS -CHO = 6.64.

[0040] Example 1 A preparation method of a long-acting high-dynamic hydrazone bond hydrogel, comprising the following steps: S1, preparing HA-Ad-ADH (M W = 58 kDa; DS -Ad = 0.17, DS -ADH = 0.21) and PEG8-BA (M W = 11 kDa, DS -CHO = 6.64) solution: 80 mg of HA-Ad-ADH and 80 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH = 7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 80 mg / mL; S2, 10 mg of βCD-mPD was dissolved in 10 mL of PBS buffer with pH = 7.4 to prepare a βCD-mPD solution with a concentration of 1 mg / mL. The βCD-mPD solution was diluted with PBS buffer to have different concentrations in turn, and the absorbance at a wavelength of 230 nm was measured by an enzyme marker. A standard curve was established as shown in Figure 6 .

[0041] S3, Preparation of long-acting high dynamic hydrazone bond hydrogel: to the HA-Ad-ADH solution prepared in step S1 (100 μL) was added βCD-mPD (0.9 mg, 0.00068 mmol), mixed uniformly, to form a host-guest assembly solution containing catalyst; then to which was added PEG8-BA solution prepared in step S1 (91 μL), stirred uniformly and incubated at room temperature or 37°C, the long-acting high dynamic hydrazone bond hydrogel (denoted as mPD@Gel1) with solid content of 8.4 w / v% was obtained by hydrazone bond coupling reaction; its storage modulus was 7200 Pa, and the concentration of catalyst βCD-mPD was 3.18 mM.

[0042] Example 2 A method for preparing a long-acting high dynamic hydrazone bond hydrogel, comprising the following steps: S1, Preparation of HA-Ad-ADH (M W =58 kDa; DS -Ad =0.17, DS -ADH =0.21) and PEG8-BA (M W =11 kDa; DS CHO =6.64) solution: 60 mg of HA-Ad-ADH and 60 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH=7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 60 mg / mL; S2, Preparation of long-acting high dynamic hydrazone bond hydrogel: to the HA-Ad-ADH solution prepared in step S1 (100 μL) was added βCD-mPD (0.9 mg, 0.00068 mmol), mixed uniformly, to form a host-guest assembly solution containing catalyst; then to which was added PEG8-BA solution prepared in step S1 (91 μL), stirred uniformly and incubated at room temperature or 20°C, the long-acting high dynamic hydrazone bond hydrogel (denoted as mPD@Gel2) with solid content of 6.5 w / v% was obtained by hydrazone bond coupling reaction; its storage modulus was 3317 Pa, and the concentration of catalyst βCD-mPD was 3.18 mM.

[0043] Example 3 A method for preparing a long-acting high dynamic hydrazone bond hydrogel, comprising the following steps: S1, Preparation of HA-Ad-ADH (M W =58 kDa; DS -Ad =0.17, DS -ADH =0.21) and PEG8-BA (M W =11 kDa; DS -CHO=6.64) Solution: Weigh 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA, and dissolve them in 1 mL of PBS buffer at pH=7.4 to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL. S2. Preparation of long-lasting, highly dynamic hydrazone hydrogel: Add βCD-mPD (0.9 mg, 0.00068 mmol) to the HA-Ad-ADH solution (100 μL) prepared in step S1, mix well to form a host-guest assembly solution containing the catalyst; then add the PEG8-BA solution (91 μL) prepared in step S1 to it, stir well and incubate at room temperature or 35°C to obtain a long-lasting, highly dynamic hydrazone hydrogel (denoted as mPD@Gel3) with a solid content of 4.5 w / v% through hydrazone coupling reaction; its storage modulus is 1533 Pa and the concentration of the catalyst βCD-mPD is 3.18 mM.

[0044] Example 4 A method for preparing a long-lasting, highly dynamic hydrazone-bonded hydrogel includes the following steps: S1. Preparation of HA-Ad-ADH (M W =58 kDa; DS -Ad =0.17, DS -ADH =0.21) and PEG8-BA (M W =11 kDa; DS -CHO =6.64) Solution: Weigh 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA, and dissolve them in 1 mL of PBS buffer at pH=7.4 to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL. S2. Preparation of long-lasting, highly dynamic hydrazone hydrogel: Add βCD-mPD (1.5 mg, 0.00113 mmol) to the HA-Ad-ADH solution (100 μL) prepared in step S1, mix well to form a host-guest assembly solution containing the catalyst; then add the PEG8-BA solution (91 μL) prepared in step S1 to it, stir well and incubate at room temperature or 30°C to obtain a long-lasting, highly dynamic hydrazone hydrogel (denoted as mPD@Gel4) with a solid content of 4.8 w / v% through hydrazone coupling reaction; its storage modulus is 1454 Pa and the concentration of the catalyst βCD-mPD is 5.44 mM.

[0045] Example 5 A method for preparing a long-lasting, highly dynamic hydrazone-bonded hydrogel, the preparation process is as follows: Figure 5 As shown, it includes the following steps: S1, preparing HA-Ad-ADH (M W = 58 kDa; DS -Ad = 0.17, DS -ADH = 0.21) and PEG8-BA (M W = 11 kDa; DS -CHO = 6.64) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH = 7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparing long-acting high-dynamic hydrazone bond hydrogel: the HA-Ad-ADH solution (100 μL) prepared in step S1 was added with βCD-mPD (2.25 mg, 0.00170 mmol), mixed uniformly to form a host-guest assembly solution containing catalyst; then the PEG8-BA solution (91 μL) prepared in step S1 was added thereto, stirred uniformly and incubated at room temperature or 33°C, so that a long-acting high-dynamic hydrazone bond hydrogel (denoted as mPD@Gel5) with a solid content of 5.2 w / v% was obtained through hydrazone bond coupling reaction; the storage modulus thereof was 1368 Pa, and the concentration of the catalyst βCD-mPD was 8.47 mM.

[0046] A schematic diagram of the reversible anchoring catalyst accelerating the exchange kinetics of the hydrazone bond in the long-acting high-dynamic hydrazone bond hydrogel in Example 5 is shown in Figure 4 .

[0047] Example 6 A method for preparing a long-acting high-dynamic hydrazone bond hydrogel, comprising the following steps: S1, preparing HA-Ad-ADH (M W = 58 kDa; DS -Ad = 0.17, DS -ADH = 0.21) and PEG8-BA (M W = 11 kDa; DS -CHO = 6.64) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH = 7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparation of long-acting high dynamic hydrazone bond hydrogel: add βCD-mPD (2.85 mg, 0.00215 mmol) to the HA-Ad-ADH solution (100 μL) prepared in step S1, mix uniformly to form a host-guest assembly solution containing a catalyst; then add the PEG8-BA solution (91 μL) prepared in step S1 thereto, stir uniformly and incubate at room temperature or 37°C, so as to obtain a long-acting high dynamic hydrazone bond hydrogel (denoted as mPD@Gel6) with a solid content of 5.5 w / v% through a hydrazone bond coupling reaction; the storage modulus thereof is 1631 Pa, and the concentration of the catalyst βCD-mPD is 10.39 mM.

[0048] Example 7 A preparation method of a long-acting high dynamic hydrazone bond hydrogel, comprising the following steps: S1, preparation of HA-Ad-ADH (M W =58 kDa; DS -Ad =0.10, DS -ADH =0.35) and PEG8-BA (M W =21 kDa; DS -CHO =5) solution: weigh 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA, respectively, and dissolve them in 1 mL of PBS buffer with pH=7.4 to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparation of long-acting high dynamic hydrazone bond hydrogel: add βCD-mPD (1.71 mg, 0.00129 mmol) to the HA-Ad-ADH solution (100 μL) prepared in step S1, mix uniformly to form a host-guest assembly solution containing a catalyst; then add the PEG8-BA solution (294 μL) prepared in step S1 thereto, stir uniformly and incubate at room temperature or 37°C, so as to obtain a long-acting high dynamic hydrazone bond hydrogel (denoted as mPD@Gel7) with a solid content of 4.4 w / v% through a hydrazone bond coupling reaction; the storage modulus thereof is 1563 Pa, and the concentration of the catalyst βCD-mPD is 3.18 mM.

[0049] Example 8 A preparation method of a long-acting high dynamic hydrazone bond hydrogel, comprising the following steps: S1, preparation of HA-Ad-ADH (M W =900 kDa; DS -Ad =0.15, DS -ADH =0.25) and PEG8-BA (M W =31 kDa; DS -CHO=7.12) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH = 7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparation of long-acting high-dynamic hydrazone bond hydrogel: the HA-Ad-ADH solution (100 μL) prepared in step S1 was added with βCD-mPD (3.08 mg, 0.00232 mmol) and mixed uniformly to form a host-guest assembly solution containing a catalyst; then the PEG8-BA solution (222 μL) prepared in step S1 was added thereto, stirred uniformly and incubated at room temperature or 37°C, so that a long-acting high-dynamic hydrazone bond hydrogel (denoted as mPD@Gel8) with a solid content of 5.0 w / v% was obtained through hydrazone bond coupling reaction; the storage modulus thereof was 1526 Pa, and the concentration of the catalyst βCD-mPD was 6.80 mM.

[0050] Example 9 A preparation method of a long-acting high-dynamic hydrazone bond hydrogel, comprising the following steps: S1, preparation of HA-Ad-ADH (M W =1729 kDa; DS -Ad =0.25, DS -ADH =0.15) and PEG8-BA (M W =41 kDa, DS -CHO =7.92) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer with pH = 7.4, respectively, to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparation of long-acting high-dynamic hydrazone bond hydrogel: the HA-Ad-ADH solution (100 μL) prepared in step S1 was added with βCD-mPD (3.08 mg, 0.00232 mmol) and mixed uniformly to form a host-guest assembly solution containing a catalyst; then the PEG8-BA solution (222 μL) prepared in step S1 was added thereto, stirred uniformly and incubated at room temperature or 37°C, so that a long-acting high-dynamic hydrazone bond hydrogel (denoted as mPD@Gel8) with a solid content of 5.0 w / v% was obtained through hydrazone bond coupling reaction; the storage modulus thereof was 1526 Pa, and the concentration of the catalyst βCD-mPD was 6.80 mM.

[0051] In order to further illustrate the technical effects of the present application, the present application also sets up a comparative example, as follows: Comparative Example 1 (blank hydrazone bond hydrogel) A preparation method of a blank hydrazone bond hydrogel, comprising the following steps: S1, preparing HA-Ad-ADH (M W = 58 kDa; DS -Ad = 0.17, DS -ADH = 0.21) and PEG8-BA (M W = 11 kDa; DS -CHO = 6.64) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer solution with pH=7.4 respectively to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, preparing a blank hydrazone bond hydrogel: the HA-Ad-ADH solution (100 μL) prepared in step S1 was mixed with the PEG8-BA solution (91 μL) uniformly and incubated at room temperature or 37°C, so that a blank hydrazone bond hydrogel (denoted as Gel0) with a solid content of 4.0 w / v% was obtained through a hydrazone bond coupling reaction; and the storage modulus thereof was 1510 Pa.

[0052] Comparative Example 2 (short-acting high-dynamic hydrazone bond hydrogel loaded with free catalyst) A preparation method of a short-acting high-dynamic hydrazone bond hydrogel loaded with free catalyst, comprising the following steps: S1, preparing HA-Ad-ADH (M W = 58 kDa; DS -Ad = 0.17, DS -ADH = 0.21) and PEG8-BA (M W = 11 kDa; DS -CHO = 6.64) solution: 40 mg of HA-Ad-ADH and 40 mg of PEG8-BA were weighed and dissolved in 1 mL of PBS buffer solution with pH=7.4 respectively to prepare HA-Ad-ADH and PEG8-BA solutions with a concentration of 40 mg / mL; S2, 10 mg of DABA was dissolved in 10 mL of PBS buffer solution with pH=7.4 to prepare a DABA solution with a concentration of 1 mg / mL, and the DABA solution with different concentrations was prepared by halving dilution with PBS buffer solution in sequence, and the absorbance of the DABA solution at a wavelength of 230 nm was measured by using an enzyme marker, so as to establish a standard curve as shown in Figure 7 .

[0053] S3, preparation of short-acting high-dynamic hydrazone bond hydrogel: to the HA-Ad-ADH solution prepared in step S1 (100 μL) was added 3,5-diaminobenzoic acid (DABA, 0.09 mg, 0.00052 mmol), mixed uniformly, then to which was added the PEG8-BA solution prepared in step S1 (91 μL), stirred uniformly and incubated at room temperature or 37 °C, a short-acting high-dynamic hydrazone bond hydrogel (denoted as DABA@Gel) with solid content of 4.1 w / v% was obtained by hydrazone bond coupling reaction; its storage modulus was 1457 Pa, and the concentration of catalyst DABA was 2.27 mM.

[0054] The performance of the hydrogels prepared in the above examples and comparative examples was evaluated, and the results are as follows: As shown in Figure 8 The solution-gel transition process of the HA-Ad-ADH / βCD-mPD pre-assembled solution after mixing with the PEG8-BA solution was recorded. In order to increase the visibility of the gel, methylene blue was used to dye the precursor solution.

[0055] Gelation time and rheological property evaluation of long-acting high-dynamic hydrazone bond hydrogel: The gelation time was determined by the inverted test tube method, and the gel precursor solution was added to the test tube and mixed, then incubated at 25 °C, and when the test tube was vertically inverted, no visible flow phenomenon occurred in the system, which was considered as gel formation. In addition, the rheological property of the hydrogel was tested at 25 °C using a rotational rheometer, the parallel plate diameter was 8 mm, and the gap between the plates was set to 1 mm. After rapidly mixing the hydrogel precursor solution on the parallel plate, a time sweep test was performed at a frequency of 1 Hz and a strain of 1% to monitor the modulus change during the formation of the hydrogel. Then a frequency sweep test was performed at a strain of 1%, and the frequency range was 0.1 Hz to 10 Hz to evaluate the viscoelastic behavior of the hydrogel. In order to evaluate the stress relaxation characteristics of the hydrogel, the stress relaxation mode scanning was performed, the shear strain was set to 10%, the decay curve of the shear stress with time was recorded, and the time required for the stress relaxation to half of the initial value was represented as τ 1 / 2 .

[0056] Table 1 is the storage and loss modulus of the hydrazone bond hydrogels with different solid contents prepared in examples 1, 2 and 3 of the present application. The results show that the storage modulus (G') of the hydrogel is significantly positively correlated with its solid content. When the solid content of the hydrogel is 4.5 w / v% (corresponding to a polymer concentration of 4 w / v %), the storage modulus value is about 1500 Pa, which can provide a stable and mechanically suitable microenvironment for stem cell culture. Therefore, in the subsequent experimental study, the polymer concentration of the hydrogel was set to 4 w / v %.

[0057] Table 1

[0058] a Concentration of HA-Ad-ADH and PEG8-BA components in the hydrogel like Figures 9-11 Table 2 shows the rheological property test results of the hydrazone hydrogels prepared in Examples 3, 4, 5, and 6 of this invention and Comparative Examples 1 and 2. Figure 9 As shown in Table 2, the gelation times of the hydrazone hydrogels in Examples 3, 4, 5, 6, and Comparative Example 2 were significantly shorter than those of the blank hydrazone hydrogel in Comparative Example 1, indicating that the catalyst can accelerate hydrazone bond formation. Furthermore, regardless of the catalyst loading, the G′ of the hydrogels in each group was approximately the same. Figure 10 As shown, all hydrazone hydrogels, including Examples 3, 4, 5, 6 and Comparative Examples 1 and 2, exhibit frequency-independent G′ values ​​in the frequency range of 0.1–10 Hz, and are consistently higher than the loss modulus (G′′), indicating that these hydrogels exhibit typical elastic solid behavior. Figure 11 As shown in Table 2, the τ values ​​of the hydrazone hydrogels in Examples 3, 4, 5, 6 and Comparative Example 2 are... 1 / 2 The significantly lower performance compared to the blank hydrazone hydrogel in Comparative Example 1 indicates that the presence of the catalyst significantly enhances the network dynamics of the hydrazone hydrogel. Given that mPD@Gel5 exhibits faster stress relaxation behavior, it was chosen for subsequent studies.

[0059] Table 2

[0060] a Concentration of HA-Ad-ADH and PEG8-BA components in the hydrogel Evaluation of the self-healing and adaptive properties of long-lasting, high-dynamic hydrazone hydrogels: Example 5: The self-healing properties of the hydrazone hydrogel prepared in Comparative Example 1 are as follows. Figure 12 As shown, two circular hydrogels stained with Rhodamine B and methylene blue, respectively, were cut in half, ensuring close contact between the two colored hemispheres along the cut surface. The healing status of the hydrogels was observed after eight hours. The test results show that the hydrazone hydrogel containing βCD-mPD (mPD@Gel5) in Example 5 can heal into a complete hydrogel. When the hydrogel is lifted with tweezers in a direction perpendicular to the cut surface, the other half of the hydrogel does not fall off even under gravity and remains intact. However, the blank hydrazone hydrogel (Gel0) of Comparative Example 1, which does not contain βCD-mPD, cannot heal itself under the same healing conditions. These results indicate that hydrazone hydrogels can undergo effective self-repair in the presence of βCD-mPD.

[0061] The self-adaptability of the hydrazone-linked hydrogel prepared in Example 5 and Comparative Example 1 was as shown Figure 13 As shown, the round hydrogel dyed with methylene blue was placed in a star-shaped mold to observe the reshaping of the hydrogel. As can be seen from the test results, the hydrazone-linked hydrogel in Example 5 added with βCD-mPD (mPD@Gel5) can be reshaped from a circle to a star, while the blank hydrazone-linked hydrogel in Comparative Example 1 without the addition of βCD-mPD (Gel0) cannot be reshaped under the same conditions. It can be seen that in the presence of βCD-mPD, the hydrazone-linked hydrogel has good structural dynamics and shape adaptability.

[0062] Evaluation of in vitro degradation performance, catalyst retention ability and rheological performance of PBS-extracted long-acting high-dynamic hydrazone-linked hydrogel: Evaluation of in vitro degradation performance: The hydrazone-linked hydrogels in Example 5 and Comparative Example 1 (200 μL) were placed in 5 mL centrifuge tubes, respectively, and after being fully matured, 2 mL of PBS buffer with pH=7.4 was added to each, and then the centrifuge tubes were incubated in a shaking bed at 37 °C and 120 rpm. At the predetermined time point, the hydrogels were taken out of the centrifuge tubes, and the remaining mass of the hydrogels was recorded, and each group of experiments was repeated three times. The experimental results are shown in Figure 14 As shown, both of them did not have obvious mass loss behavior after being soaked in PBS for 168 h, indicating that the hydrazone-linked hydrogel has good hydrolysis resistance and can maintain structural and mechanical stability, providing a stable three-dimensional microenvironment for stem cell culture.

[0063] Evaluation of catalyst retention ability: The hydrazone-linked hydrogels in Example 5 and Comparative Example 2 (200 μL) were placed in 5 mL centrifuge tubes, respectively, and after being fully matured, 2 mL of PBS buffer with pH=7.4 was added to each, and then the centrifuge tubes were incubated in a shaking bed at 37 °C and 120 rpm. At the predetermined time point, 2 mL of supernatant was taken out of the centrifuge tubes, and 2 mL of fresh buffer was supplemented. The absorbance of the supernatant at 230 nm was measured, and the cumulative release rate was calculated according to the standard curve of βCD-mPD and DABA, and each group of experiments was repeated three times. The test results are shown in Figure 15 As shown, the free catalyst DABA can be released from the hydrogel by nearly 100% within 2 h, while the anchored catalyst βCD-mPD is only released by 25% within 48 h, indicating that the host-guest complexation can significantly enhance the retention ability of the catalyst in the gel network. This excellent catalyst retention performance is conducive to maintaining the long-term structural dynamics of the hydrogel, thereby providing a sustained dynamic microenvironment for stem cell culture.

[0064] Rheological property evaluation of hydrogels after PBS immersion: The hydrazone-linked hydrogels (200 μL) in Example 5 and Comparative Example 2 were placed in 5 mL centrifuge tubes, and after being fully matured, 2 mL of PBS buffer at pH = 7.4 was added, and then the centrifuge tubes were incubated in a shaking incubator at 37 °C and 120 rpm for 48 h. The hydrogels were taken out and subjected to frequency sweep and stress relaxation tests using a rotational rheometer. The test results are shown in Figure 16 Fig. 2. As shown in Figure 16 Fig. 2a, after 48 h of immersion in PBS, the G' of both the DABA@Gel and mPD@Gel5 hydrogels decreased slightly, but still remained at about 1000 Pa, meeting the requirements of stem cell culture on mechanical properties. As shown in Figure 16 Fig. 2b, after 48 h of immersion in PBS, the τ 1 / 2 was greatly extended from 404 s before immersion to 3107 s, and the structural dynamics was almost lost; in contrast, the τ 1 / 2 of the mPD@Gel5 hydrogel was slightly extended from 448 s before immersion to 516 s, and still maintained good structural dynamics. The above results show that the mPD@Gel5 hydrogel has good structural stability and long-term dynamics in a physiological environment, and can provide sustained stable mechanical support and dynamic microenvironment for stem cells.

[0065] Biological safety evaluation of long-acting and high-dynamic hydrazone-linked hydrogels: Blood compatibility test: 500 μL of mouse whole blood was taken into an anticoagulant tube, and then dispersed into 5 mL of physiological saline. Red blood cells were collected by multiple centrifugations until the supernatant was clear and transparent, and then resuspended with physiological saline. 500 μL of red blood cell suspension was mixed with 500 μL of different concentrations of DABA, βCD-mPD and mPD@Gel5 hydrogel immersion liquid in Example 5 (200 mg of hydrazone-linked hydrogel was immersed in 1 mL of physiological saline for 48 h to obtain the hydrogel immersion liquid, which was then diluted step by step for use), and at the same time, 500 μL of red blood cell suspension + 500 μL of physiological saline was used as a negative control group, and 500 μL of red blood cell suspension + 500 μL of deionized water was used as a positive control group. They were incubated in a shaking incubator at 37 °C and 120 rpm for 1 h, and the supernatant was collected by centrifugation. The absorbance at 540 nm wavelength was measured to calculate the hemolysis rate. Each group of experiments was repeated three times.

[0066] Cell compatibility test: The toxicity of different concentrations of DABA, βCD-mPD and mPD@Gel5 hydrogel in Example 5 on cells was evaluated by MTT experiment. Specifically, human bone marrow mesenchymal stem cells (hMSCs) were seeded in 96-well plates at a density of 5 × 10 4The cells were seeded at a density of 1 cell / well in 96-well plates containing α-MEM complete medium (containing 17% fetal bovine serum, 1% penicillin-streptomycin, 1% L-glutamine and 5 ng / mL recombinant human fibroblast growth factor 2) and incubated at 37°C in a sterile incubator with 5% CO2for 12 h to allow the cells to grow completely adherent. The old medium was removed and α-MEM complete medium containing different concentrations of DABA, βCD-mPD and hydrogel extract was added. The cells cultured with pure α-MEM complete medium were used as a positive control, and pure α-MEM complete medium was used as a negative control. The cells were incubated for another 48 h under the same culture conditions. 20 μL of MTT solution (5 mg / mL) was added to each well of the 96-well plate, and the incubation was continued for another 4 h. The MTT solution was removed, 200 μL of DMSO was added to each well, and the absorbance at 570 nm was measured after 15 min. The cell survival rate was calculated.

[0067] As shown in Figures 17-19 The results of the biological safety evaluation of DABA, βCD-mPD and mPD@Gel5 hydrogel prepared in the present application are shown. As shown in Figure 17 and Figure 18 The hemolysis experiment results show that when the concentration of DABA reaches 1250 μg / mL, obvious hemolysis occurs, while βCD-mPD and mPD@Gel5 hydrogel still have no hemolysis phenomenon when the concentration reaches 5000 μg / mL and 200 mg / mL respectively, and the hemolysis rate is less than 5%, indicating that βCD-mPD and mPD@Gel5 have good blood compatibility. As shown in Figure 19 The results of the cytotoxicity experiment show that when the concentration of DABA reaches 625 μg / mL, obvious cytotoxicity occurs, while the cell survival rate of βCD-mPD and mPD@Gel5 hydrogel is more than 80% when the concentration reaches 5000 μg / mL and 200 mg / mL respectively, indicating that βCD-mPD and mPD@Gel5 have good cell compatibility. The above results together show that the catalyst βCD-mPD and the mPD@Gel5 hydrogel prepared therefrom have good biological safety.

[0068] Evaluation of the three-dimensional culture of hMSCs and the in vitro osteogenic differentiation ability of long-acting high-dynamic hydrazone bond hydrogel: To facilitate cell adhesion to the hydrogel matrix, RGD-functionalized HA-Ad-ADH (HA-Ad-ADH-RGD) was prepared by covalently linking RGD peptides to the hydrazide groups of HA-Ad-ADH via amide condensation reaction. The detailed procedure was as follows: Acrylated HA-Ad-ADH was first prepared. HA-Ad-ADH (1 g, 0.42 mmol-ADH) was dissolved in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer at pH 7.2, and N-acryloyloxy succinimide (11.84 mg, 0.07 mmol) was added. The reaction was stirred at room temperature overnight. The product was purified by dialysis against deionized water and then freeze-dried to obtain acrylated HA-Ad-ADH (HA-Ad-ADH-AC). Subsequently, HA-Ad-ADH-AC (200 mg, 0.4 mmol) was dissolved in 10 mL phosphate buffer (0.2 M, pH 8.0) and stirred at room temperature for 2 h. Then, thiol-functionalized RGD peptides (GCGYGRGDSPG, 246.04 mg, 0.48 mmol) were added to the above solution. Finally, tris(2-carboxyethyl)phosphine hydrochloride (170.13 mg, 0.68 mmol) was added and the reaction was stirred at room temperature for 20 h. The product was purified by dialysis against deionized water and then freeze-dried to obtain HA-Ad-ADH-RGD.

[0069] hMSCs were encapsulated into mPD@Gel5 hydrogels of Example 5 and DABA@Gel hydrogels of Comparative Example 2 under sterile conditions and cultured in three dimensions. The detailed procedure was as follows: PEG8-BA and HA-Ad-ADH-RGD solutions were prepared with a concentration of 40 mg / mL in PBS, respectively. For the mPD@Gel5 hydrogel group, 278 μL of HA-Ad-ADH-RGD solution was mixed with 5.7 mg of βCD-mPD to obtain a HA-Ad-ADH-RGD / βCD-mPD complex solution. Then, hMSCs were uniformly dispersed in the above complex solution to form a cell suspension. Subsequently, the cell suspension and 222 μL of PEG8-BA solution were added to a 24-well plate, stirred with a pipette tip, and incubated in a 37°C incubator for 10 min to form mPD@Gel5 hydrogel / cell complexes (5 x 105cells mL-1). The DABA@Gel hydrogel group was prepared in the same way as the mPD@Gel5 hydrogel group, except that βCD-mPD was replaced by DABA. 5 cells mL -1). For the DABA@Gel hydrogel group, 273 μL of HA-Ad-ADH-RGD solution was mixed with 0.78 mg of DABA to prepare a HA-Ad-ADH-RGD / DABA complex solution. Then, hMSCs were uniformly dispersed in the above complex solution to form a cell suspension. Subsequently, the cell suspension and 227 μL of PEG8-BA solution were added to a 24-well plate, mixed uniformly with a pipette tip, and incubated in a 37°C incubator for 10 min to form a DABA@Gel hydrogel / cell complex (5 x 105cells mL 5 cells mL -1 ). Then, 2 mL of α-MEM medium was added to each well, and the plate was transferred to a 37°C, 5% CO2incubator. The medium was changed for the first time after 1 h, and then every 12 h. After 48 h of culture, the medium was discarded, and the hydrogel / cell complex was fixed in 4% paraformaldehyde at room temperature for 1 h, washed with DPBS for 3 times, and permeabilized with 0.1 v / v% Triton X-100 for 30 min. Subsequently, the hydrogel / cell complex was incubated in a 50 mg / mL BSA solution for 30 min to block non-specific binding. After the BSA solution was discarded, a 50 mg / mL BSA solution containing anti-mechanical sensitivity transcription factor YAP primary antibody (dilution ratio 1:200) was added, and the plate was incubated at 4°C overnight. The next day, the hydrogel / cell complex was washed with 5 μL / mL Tween-20 / PBS solution for 3 times. Then, goat anti-mouse Alexa Fluor® 488 labeled secondary antibody (dilution ratio 1:200), phalloidin (dilution ratio 1:200), and 4',6-diamidino-2-phenylindole (DAPI, 0.1 μg / mL) were added, and the plate was incubated at room temperature in the dark for 90 min to label YAP protein, F-actin, and the nucleus, respectively. Finally, the plate was washed with 5 μL / mL Tween-20 / PBS solution for 5 times, and the samples were stored in DPBS before imaging. Fluorescence images were collected using a confocal microscope to observe the morphological changes of hMSCs in the hydrogel and the nuclear translocation of YAP protein.

[0070] Following the above method, 50 μL of mPD@Gel5 hydrogel / cell complex and DABA@Gel hydrogel / cell complex were prepared in 96-well plates. Then, 200 μL of osteogenic induction medium (containing 10 mM β-glycerophosphate disodium, 50 μg / mL L-ascorbic acid, and 100 nM dexamethasone) was added to each well, and the plates were cultured for 7 days, with the induction medium changed every 2 days. After culture, samples were collected and homogenized using TRIzol reagent, and total RNA was extracted according to the manufacturer's instructions. RNA concentration was then measured using a spectrophotometer. 100 ng of RNA from each sample was reverse transcribed into cDNA using the RevertAid first-strand cDNA synthesis kit. Real-time quantitative polymerase chain reaction (RT-qPCR) was then performed to detect the expression levels of osteogenic marker genes (Runt-related transcription factor 2, Runx 2; alkaline phosphatase, ALP; type I collagen, Col I; osteocalcin, OCN), with GAPDH as an internal control gene. Relative gene expression levels were calculated using the ΔΔCt method, with fold change values ​​expressed as 2. ΔΔCt The formula is used to calculate this.

[0071] like Figure 20 The image shows F-actin immunofluorescence staining images of hMSCs after 48 h of culture in mPD@Gel5 and DABA@Gel hydrogels. Notably, compared to the round and unspread cell morphology observed in DABA@Gel hydrogel, the cells in mPD@Gel5 hydrogel formed extensive star-shaped spread and interconnected cell networks. Figure 21 The image shows YAP immunofluorescence staining images of hMSCs after 48 h of culture in mPD@Gel5 and DABA@Gel hydrogels. Compared with DABA@Gel hydrogel, nuclear translocation of YAP was more significant in mPD@Gel5 hydrogel. This is attributed to the highly spread cells in mPD@Gel5 causing nuclear pores to expand under tension, thereby reducing the steric hindrance of YAP into the nucleus. These results indicate that, compared with DABA@Gel hydrogel, mPD@Gel5 hydrogel can maintain higher network dynamics during cell culture, thereby promoting cell spread and activating mechanosensitive signaling pathways.

[0072] Since YAP nucleation can upregulate the expression of osteogenic-related genes such as Runx 2, we further evaluated the mechanosensitive-dependent osteogenic capacity of hMSCs encapsulated in two hydrogels. Results are as follows: Figure 22As shown, after 7 days of culture in osteogenic induction medium, cells in mPD@Gel5 hydrogel showed a significant up-regulation of osteogenic marker gene expression levels compared with hMSCs in DABA@Gel hydrogel. It can be seen that the long-acting and high-dynamic cross-linking characteristics of mPD@Gel5 hydrogel endow the network with good remoldability, providing a structural basis for cell star-shaped spreading; at the same time, the highly spread cell morphology can promote efficient nuclear import of YAP and up-regulate the expression of osteogenic markers such as Runx 2, ALP, etc. In contrast, DABA@Gel hydrogel is difficult to maintain high dynamicity during cell culture, and cannot effectively transmit mechanical signals, so its osteogenic differentiation ability is weak.

[0073] In summary, the long-acting and high-dynamic hydrazone bond hydrogel prepared by the present application effectively solves the problem that existing hydrogels cannot balance micro-dynamicity and macro-stability by reasonably designing the carrier raw material and catalyst anchoring mode. This material not only has excellent biocompatibility, but also can provide sustained and stable mechanical support and dynamic microenvironment for stem cells to support stem cell spreading, activate mechanical signal transduction, promote osteogenic-related gene expression, and enhance the osteogenic differentiation ability of stem cells, and has important application value in the field of stem cell culture platform construction.

[0074] The above examples are only used to illustrate the technical solutions of the present application, but not limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing long-acting high dynamic hydrazone bond hydrogel, characterized in that, The method comprises the following steps: The long-acting high-dynamic hydrazone bond hydrogel is prepared by mixing the aqueous solution of sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide with the m-phenylenediamine modified by β-cyclodextrin, adding the aqueous solution of benzaldehyde-terminated eight-arm polyethylene glycol, and incubating. The structural formula of the β-cyclodextrin modified m-phenylenediamine is: .

2. The production method according to claim 1, characterized by, The preparation method of the m-phenylenediamine modified by β-cyclodextrin is as follows: First, the β-cyclodextrin (βCD) is reacted with p-toluenesulfonyl chloride, and then reacted with 1,3-propanediamine to obtain mono-(6-propanediamine-6-deoxy)-βCD; the tert-butyloxycarbonyl-protected 3,5-diaminobenzoic acid is bonded to the mono-(6-propanediamine-6-deoxy)-βCD through an amide condensation reaction; and the tert-butyloxycarbonyl protecting group is removed using trifluoroacetic acid.

3. The preparation method according to claim 1, characterized in that, The molar ratio of β-cyclodextrin modified m-phenylenediamine to 1-adamantane acetic acid is 1:0.8-1:2, and the molar ratio of benzaldehyde-terminated eight-arm polyethylene glycol aldehyde group to adipic acid dihydrazide hydrazine group is 1:1-1:2; the substitution degree of 1-adamantane acetic acid and adipic acid dihydrazide modified sodium hyaluronate is DS -Ad =0.10-0.25, and DS -ADH =0.15-0.35, respectively, in terms of the number of moles of 1-adamantane acetic acid and adipic acid dihydrazide on 1 mole of disaccharide repeat unit.

4. The method of claim 1, wherein, The weight average molecular weight of the sodium hyaluronate modified by 1-adamantane acetic acid and adipic acid dihydrazide is 58 kDa-1729 kDa.

5. The preparation method according to claim 1, characterized in that, The degree of substitution, DS, of the benzaldehyde-terminated eight-arm polyethylene glycol is calculated as the number of aldehyde groups on the benzaldehyde-terminated eight-arm polyethylene glycol molecule per benzaldehyde end group -CHO = 5-7.

92.

6. The method of claim 1, wherein, The weight average molecular weight of the benzaldehyde-terminated eight-arm polyethylene glycol is 11 kDa-41 kDa.

7. The preparation method according to claim 1, characterized in that, The concentration of the m-phenylenediamine modified by β-cyclodextrin in the reaction solution is 3.18-10.39 mM; and the solid content of the long-acting high-dynamic hydrazone bond hydrogel is 4.5 w / v%-8.4 w / v%.

8. The method of claim 1, wherein, The incubation temperature is 20-37℃, and the incubation time is 10-20 min.

9. The long-acting high-dynamic hydrazone bond hydrogel prepared by the preparation method of any one of claims 1-8.

10. The long-acting high-dynamic hydrazone bond hydrogel of claim 9 is used in three-dimensional culture of stem cells.

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