Dynamic crosslinking supramolecular hydrogel and preparation method thereof

Abstract

The invention relates to supramolecular hydrogel formed through subject-object interaction and Schiff base bond dynamic crosslinking and a preparation method of the supramolecular hydrogel. The hydrogel is formed by self-assembly of biological macromolecules modified by hydrophobic groups, cyclodextrin modified by active carbonyl groups and biological macromolecules modified by amino or hydrazide groups, and the biological macromolecules serve as basic materials of the hydrogel and are selected from hyaluronic acid, gelatin, chitosan, glucan, pectin or carboxymethyl cellulose. The preparation method of the hydrogel comprises the following steps: mixing hydrophobic group modified biomacromolecules and active carbonyl modified cyclodextrin to prepare an aqueous solution A; preparing an aqueous solution B from the amino or hydrazide group modified biological macromolecules; and mixing the aqueous solution A and the aqueous solution B to obtain the dynamic crosslinking supramolecular hydrogel. The supramolecular hydrogel disclosed by the invention has self-healing property and shear thinning property, meanwhile, the plasticity is enhanced, and cell migration, proliferation and differentiation can be promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical polymer materials, and particularly relates to a dynamically crosslinked supramolecular hydrogel, a preparation method thereof, and an application thereof. Background Art

[0002] Hydrogel is a material similar to the extracellular matrix and is widely regarded as a promising scaffold material in tissue engineering and regenerative medicine. The traditional hydrogel network forms a covalently crosslinked three-dimensional network through light irradiation, that is, free radicals are generated by photoinitiation, and then a highly hydrated three-dimensional covalent network is formed by free radical polymerization. The hydrogel constructed in this way has a stable chemical structure and good mechanical properties; however, due to the irreversibility of chemical crosslinking, it cannot self-heal and is not easily degraded, which cannot meet the special needs of cell culture. Supramolecular hydrogel refers to a three-dimensional hydrogel constructed through physical and chemical interactions, including hydrogen bonds, host-guest interactions, dynamic ion coordination interactions, dynamic covalent interactions, hydrophilic-hydrophobic interactions, etc. The three-dimensional hydrogel constructed through physical and chemical interactions generally has good mechanical properties (dynamicity, anti-swelling, etc.), that is, the hydrogel network can maintain stability during the culture process, and the dynamicity endows a certain degree of fluidity and shape adaptability, which has an important regulatory and supporting effect on cell behavior. In addition, supramolecular hydrogel has biophysical properties very similar to natural tissues, so it can be used as an efficient 3D cell culture matrix.

[0003] Currently, the methods for constructing hydrogel networks are mostly photocuring and rapid self-crosslinking. The hydrogels constructed by these methods may have a certain impact during the encapsulation process. Therefore, exploring new gelation methods is of great significance for applications in regenerative medicine, disease research, drug screening, etc. The degradation of hydrogels is also the key point of three-dimensional culture. The existing hydrogel systems require a large amount of enzymes for degradation and have low degradation efficiency, which cannot well meet the needs.

[0004] In addition, the common materials for three-dimensional culture are mostly matrix gel, natural self-crosslinked hydrogel, and covalently crosslinked hydrogel. These materials have certain defects. For example, the composition of matrix gel is unclear, the natural self-crosslinked hydrogel requires a large amount of enzymes for degradation and has a long degradation time, and the covalently self-crosslinked hydrogel can hardly be degraded, which cannot meet the requirement of rapid and gentle degradation after culturing for a certain period of time. Another problem is that most of the existing materials construct hydrogel structures through light crosslinking. Free radicals will be generated during the light irradiation process, and the free radicals may cause damage to cells, which is not conducive to the subsequent cell culture. Summary of the Invention

[0005] In view of the problems existing in the above-mentioned prior art, one of the objectives of the present invention is to provide a new supramolecular hydrogel formed by host-guest interaction and dynamic cross-linking of Schiff base bonds. The hydrogel is self-assembled from a biomacromolecule modified with a hydrophobic group, a cyclodextrin modified with a reactive carbonyl group, and a biomacromolecule modified with an amino or hydrazide group, wherein the biomacromolecule, as the basic material of the hydrogel, can be hyaluronic acid, gelatin, chitosan, dextran, pectin or carboxymethyl cellulose.

[0006] In certain embodiments, the hydrophobic group is selected from one or more of adamantane, tert-butylbenzene, acetylsalicylic acid, ibuprofen, menthol, geraniol, and cholic acid.

[0007] In certain embodiments, the reactive carbonyl in the cyclodextrin modified with a reactive carbonyl group is an aldehyde group; in certain embodiments, the amino group is hexanediamine, and the hydrazide group is adipic dihydrazide or succinic dihydrazide.

[0008] Another objective of the present invention is to provide a method for preparing a dynamically cross-linked supramolecular hydrogel, comprising the following steps:

[0009] (1) Prepare a biomacromolecule modified with a hydrophobic group, a cyclodextrin modified with a reactive carbonyl group, and

[0010] a biomacromolecule modified with an amino or hydrazide group respectively; wherein the biomacromolecule is selected from hyaluronic acid, gelatin, chitosan, dextran, pectin, and carboxymethyl cellulose,

[0011] (2) Mix the biomacromolecule modified with a hydrophobic group and the cyclodextrin modified with a reactive carbonyl group to prepare aqueous solution A; prepare aqueous solution B from the biomacromolecule modified with an amino or hydrazide group;

[0012] (3) Mix aqueous solutions A and B to obtain a hydrogel.

[0013] In certain embodiments, the hydrophobic group is selected from adamantane, tert-butylbenzene, acetylsalicylic acid, ibuprofen, menthol, geraniol, and cholic acid;

[0014] In certain embodiments, the reactive carbonyl is an aldehyde group, or a monoaldehyde compound such as acrolein dimethyl acetal or aminoacetaldehyde dimethyl acetal.

[0015] In certain embodiments, the hydrazide group is selected from adipic dihydrazide or succinic dihydrazide.

[0016] In certain embodiments, in step 2), the preparation of aqueous solution A is to mix the biomacromolecule modified with a hydrophobic group and the cyclodextrin modified with a reactive carbonyl group and dissolve them in water, a buffer solution, or a physiological solution, etc.; the preparation of aqueous solution B is to dissolve the biomacromolecule modified with an amino or hydrazide group in water, a buffer solution, or a physiological solution, etc.

[0017] The present invention further provides a method for culturing cells in a dynamic cross-linked hydrogel in vitro, comprising the following steps: mixing a biomacromolecule modified with a hydrophobic group and a cyclodextrin modified with an active carbonyl group to prepare an aqueous solution A1; dissolving a biomacromolecule modified with an amino or hydrazide group in water or a buffer solution, adding cells and mixing evenly to prepare an aqueous solution B1; mixing the aqueous solution A1 and the aqueous solution B1, adding a cell culture medium after obtaining the hydrogel for three-dimensional culture.

[0018] The present invention also relates to the application of a new dynamic cross-linked hydrogel in three-dimensional cell culture, drug delivery, preparation of injectable agents and tissue engineering.

[0019] In order to ensure the biocompatibility of the carrier, the present invention selects biomedicinal macromolecules and cyclodextrin as the base materials, which have good biocompatibility and strong hydrophilicity and have been clinically applied. After modifying the biomedicinal macromolecules and cyclodextrin, supramolecular hydrogels are constructed by self-assembly. The Schiff base interaction has pH responsiveness. The hydrogel can be degraded by adjusting the pH, and can also be degraded by adding adamantylamine or glycine to release cells and cell spheres from the hydrogel and re-encapsulate them into a new hydrogel, meeting the three-dimensional culture model for application in regenerative medicine, disease research and drug screening.

[0020] In this application, by connecting the host-guest interaction and the Schiff base bond in series, the prepared hydrogel has excellent physical and chemical properties. Combining the host-guest interaction and the Schiff base bond changes its mechanical behavior, making it more plastic. Plasticity can promote cell migration, proliferation and differentiation. The plastic hydrogel can be widely applied in three-dimensional culture, which is of great significance for clinical applications and has good application prospects in the field of biotechnology. Further, through research, it is found that by modifying aldehyde groups into cyclodextrin, the cyclodextrin first forms a host-guest interaction with the guest molecule modified on hyaluronic acid, and then the aldehyde group and the hydrazide group on the cyclodextrin are self-assembled to form a Schiff base bond, so as to ensure that the supramolecular hydrogel constructed by connecting the host-guest interaction and the Schiff base bond in series has outstanding technical effects. Description of the Drawings

[0021] Figure 1 a) 1H NMR spectrum of HA-TP (D2O); b) 1H NMR spectrum of HA-ADH (D2O); c) 1H NMR spectrum of β-CD-ald (deuterated DMSO); d) 1H NMR spectrum of carboxylated β-CD-ald 1 H NMR spectrum (deuterated DMSO); d) carboxylated β-CD-ald 1 H NMR spectrum (D2O).

[0022] Figure 2A) Schematic diagram of the gelation of SHG hydrogel; B) Demonstration of injectability, C) Alternating strain sweep storage modulus G' and loss modulus G'' at room temperature; D) Viscosity change with shear rate.

[0023] Figure 3 a) FT-IR spectrum of SHG hydrogel and b) 1 1H nuclear magnetic resonance spectrum.

[0024] Figure 4 a) Strain-time curves; b) Permanent strain of SHG hydrogel, HG hydrogel and S hydrogel.

[0025] Figure 5 a) Light microscopy images of hydrogels for culturing 143-B cells; b) Diameter of three-dimensional tumor spheres cultured in hydrogels for 7 days.

[0026] Figure 6 Light microscopy image (10X) of SHG hydrogel for culturing HepG2 cells.

[0027] Figure 7 Live / dead staining results of HepG2 cells cultured in hydrogels for 7 days.

[0028] Figure 8 a) Immunostaining of the stemness marker SOX2 for HepG2 cell spheres cultured in SHG hydrogels (40X); b) Immunostaining of the stemness marker Nanog for HepG2 cell spheres cultured in SHG hydrogels (40X); c) Statistical analysis of the relative fluorescence intensities of SOX2 and Nanog.

[0029] Figure 9 1H nuclear magnetic resonance spectrum of HA-TP-RGD.

[0030] Figure 10 a) Light microscopy image (10X) of SHG hydrogel for culturing MSCs; b) Live / dead staining (10X) of MSCs cultured in hydrogels for 7 days; c) Immunofluorescence staining images (20X) of MSCs cultured in hydrogels for 7 days. Detailed implementation manners

[0031] The reagents used in the embodiments of this application are all commercially available products. The sources of HepG2 cells, MSCB cells, and B-143 cells: Shanghai Institute of Cell Biology, Chinese Academy of Sciences.

[0032] In some embodiments, this application provides a method for preparing the hydrogel, including the steps:

[0033] (1) Prepare biomacromolecules modified with hydrophobic groups, cyclodextrins modified with active carbonyl groups, and biomacromolecules modified with hydrazide groups respectively; wherein the biomacromolecules are selected from gelatin, chitosan, dextran, pectin, and carboxymethyl cellulose;

[0034] (2) Mix the biomacromolecules modified with hydrophobic groups and the cyclodextrins modified with active carbonyl groups to prepare aqueous solution A; prepare aqueous solution B with the biomacromolecules modified with hydrazide groups;

[0035] (3) Mix aqueous solutions A and B and let it stand to obtain a hydrogel.

[0036] In some embodiments, the biomacromolecule is hyaluronic acid. The preparation method of the hyaluronic acid modified with hydrophobic groups in the above step (1) includes:

[0037] a1. Stir and dissolve sodium hyaluronate (HA-Na) powder in deionized water at room temperature, add cation exchange resin, stir, centrifuge, and filter off the resin to obtain a hyaluronic acid aqueous solution with pH = 2 - 3; stir and dropwise add tetrabutylammonium hydroxide (TBA) to the above aqueous solution until pH = 7.0. Lyophilize the above neutralized solution to obtain HA-TBA;

[0038] b1. Add HA-TBA into a round-bottom flask, dissolve it completely with DMSO, then add p-tert-butylphenylacetic acid (TP) and 4-dimethylaminopyridine (DMAP), stir to dissolve, and add di-tert-butyl dicarbonate (BOC2O) at 45 °C, and stir to react;

[0039] c1. After the reaction, dialyze and lyophilize to obtain p-tert-butylphenylacetic acid modified hyaluronic acid (HA-TP).

[0040] The dissolution concentration of the hyaluronic acid powder is 1% - 2%, the dissolution concentration of HA-TBA in anhydrous DMSO is 0.5% - 1.5%, and the dissolution temperature is 40 - 60 °C. In some embodiments, the molar ratio of HA-TBA, TP, DMAP, and BOC2O is 1:(2 - 4):(0.5 - 1):(1 - 1.2).

[0041] In some embodiments, the preparation method of the hyaluronic acid modified with hydrazide groups is:

[0042] a2. Dissolve hyaluronic acid in deionized water, add the activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), and stir and catalyze at room temperature; wherein, the dissolution concentration of the HA in deionized water is 0.1% - 1%.

[0043] b2. Add adipic dihydrazide (ADH) monomer to the above solution, stir and react at room temperature, maintain the pH value of the solution neutral during stirring, and the reaction time at room temperature is 6-8 h;

[0044] c2. Wait until the reaction proceeds to a certain extent, dialyze the reaction solution, and freeze-dry after complete dialysis to obtain a white flocculent substance, which is hyaluronic acid modified with adipic dihydrazide (HA-ADH).

[0045] In some embodiments, the molar ratio of HA, EDC, HOBt, and ADH is 1:(4.5-5):(5-5.5):(9-11).

[0046] In some embodiments, the preparation method of the active carbonyl group-modified cyclodextrin in step (1) is as follows:

[0047] a3. Dissolve β-CD in anhydrous DMSO.

[0048] b3. Dissolve Dess Martin periodate in anhydrous DMSO, add it to the above β-CD solution, and stir and react at room temperature.

[0049] c3. Drop the reaction solution into cold acetone, filter, and obtain the final product β-CD-ald.

[0050] In some embodiments, the concentration of β-CD dissolved in anhydrous DMSO is 0.05%-0.1%; the molar ratio of β-CD to Dess Martin periodate is 1:(2.9-3.1).

[0051] In some embodiments, dissolve the above-prepared HA-TP and β-CD-ald together in PBS solution, name it solution A, dissolve the HA-ADH freeze-dried flocculent substance in PBS solution, name it solution B, mix solution A and solution B, stir evenly, and let it stand at room temperature to form a gel. The gel is denoted as SHG hydrogel. Among them, the concentration ratio (w / v) of HA-TP, β-CD-ald, and HA-ADH is 1:(0.8-1.2):(1.5-2.5), and the solid content of the HA-TP precursor solution for gel formation is not less than 1%. In some embodiments, the volume ratio of solution A to solution B is 1:1. The standing time at room temperature is 1-60 min.

[0052] Example 1

[0053] The embodiment of the present application prepared a hydrogel formed by self-assembly of tandem dynamic cross-linking bonds, which includes hyaluronic acid modified with hydrophobic groups, hyaluronic acid modified with adipic dihydrazide, and cyclodextrin modified with aldehyde groups. The relevant preparation chemical reactions are shown below.

[0054]

[0055] 1) Preparation of tert - butylphenylacetic acid - esterified hyaluronic acid (HA - TP)

[0056] Sodium hyaluronate (HA - Na) powder was stirred and dissolved in deionized water at room temperature to prepare a 2% aqueous hyaluronic acid solution. Cation exchange resin was added, and the mixture was stirred at room temperature, centrifuged, and the resin was filtered off. After testing the pH of the solution, the next step could be carried out within the pH range of 2 - 3.

[0057] Dowex 50W resin with a mass three times that of sodium hyaluronate was added to the sodium hyaluronate aqueous solution, and the mixture was stirred at room temperature for 8 hours. The aqueous solution - resin mixture was placed in a centrifuge and centrifuged at 4000 rpm to preliminarily remove the resin, and the centrifuged residual liquid was temporarily stored. The obtained supernatant solution was filtered through a Buchner funnel to further remove the resin, obtaining a hyaluronic acid solution with pH = 2 - 3. The hyaluronic acid solution was adjusted to pH = 7.0 with aqueous tetrabutylammonium hydroxide to obtain the tetrabutylammonium salt of hyaluronic acid HA - TBA. After freeze - drying, a white foam of HA - TBA was obtained. 1 g of HA - TBA was weighed into a 250 - mL round - bottom flask, 100 mL of anhydrous DMSO was added, and the mixture was stirred and dissolved at 45°C. 1.616 g of p - tert - butylphenylacetic acid and 260 mg of 4 - dimethylaminopyridine were successively added to the flask where HA - TBA was located, and the mixture was stirred at room temperature for 20 min. After complete dissolution, 642 μL of BOC2O was added, and the reaction was carried out in a 45°C water bath for 24 hours. It was loaded into a dialysis bag with a molecular weight cut - off of 3500 kDa and dialyzed with DMSO dialysis solution for 2 days, with DMSO replaced once a day. Then it was dialyzed with brine for 1 day, with brine replaced three times a day. Finally, it was dialyzed with deionized water for 3 days, with deionized water replaced three times a day, and HA - TP was obtained after freeze - drying.

[0058] 2) Preparation of adipic dihydrazide - modified hyaluronic acid (HA - ADH)

[0059] 1 g of hyaluronic acid foam was weighed into a three - necked round - bottom flask containing 200 mL of deionized water and stirred and dissolved at room temperature. After complete dissolution, 2.396 g of N - (3 - dimethylaminopropyl) - N′ - ethylcarbodiimide hydrochloride and 1.914 g of 1 - hydroxybenzotriazole were added and catalyzed for 10 min. Then 4.6 g of adipic dihydrazide was added, and the mixture was stirred at room temperature for 6 h. During the stirring process, NaOH solution or hydrochloric acid solution was continuously added to the solution to maintain the solution at pH = 6.8. After the reaction, it was dialyzed with brine for 3 days, with brine replaced three times a day. Finally, it was dialyzed with deionized water for 3 days, with deionized water replaced three times a day, and HA - ADH was obtained after freeze - drying.

[0060] 3) Preparation of functionalized cyclodextrin

[0061] 3 g of β-CD was vacuum-dried for 24 h and then dissolved in 35 mL of anhydrous DMSO under nitrogen protection. 2.239 g of Dess Martin periodate was dissolved in 45 mL of anhydrous DMSO, and this solution was added dropwise to the above reaction solution within 1 h. After the titration was completed, the mixture was stirred for another 1 h. Finally, the reaction solution was added dropwise to 500 mL of cold acetone for precipitation, and the crude product was obtained by filtration; the crude product was dissolved in 60 mL of deionized water and again added dropwise to 500 mL of cold acetone for precipitation. The precipitate was filtered and dried in a vacuum drying oven at 50 °C for 12 h to obtain dry β-CD-ald, which was ground to obtain powdered β-CD-ald.

[0062] The above synthesized compounds were characterized by 1 1H nuclear magnetic resonance to confirm the successful grafting of TP and ADH and the successful oxidation of β-CD.

[0063] As Figure 1 shown, from the 1 1H nuclear magnetic resonance of HA-ADH, the methylene protons of ADH were at 1.53 ppm, and the methyl protons of N-acetylglucosamine in the HA backbone were at 1.98 ppm. The spectrum confirmed that ADH had been modified onto hyaluronic acid. From the 1 1H nuclear magnetic resonance of HA-TP, the hydrogens on the benzene ring of TP at 7.50 and 7.34 ppm and the hydrogens in the HA backbone at 3.1 - 4.1 ppm indicated that TP had been modified onto hyaluronic acid. From the 1 1H nuclear magnetic resonance of β-CD-ald, the aldehyde matrix protons were at 9.69 ppm and the primary hydroxyl groups of the cyclodextrin host at 4.82 ppm were oxidized to aldehyde groups.

[0064] 4) Gelation experiment

[0065] The above-prepared HA-TP solid and powdered β-CD-ald were co-dissolved in PBS solution at concentrations in the range of 1% - 2.5% (w / v), named solution A. The HA-ADH freeze-dried product was dissolved in PBS solution at a concentration in the range of 1% - 2.5% (w / v), named solution B. Solution A and solution B were mixed in a ratio of 1:1, stirred evenly, and allowed to gel at room temperature to obtain the SHG hydrogel. The gelation time was negatively correlated with, for example, the concentration. As the concentrations of solution A and solution B increased, the gelation time gradually shortened.

[0066] Among them, the concentration ratio (w / v) of HA-TP, β-CD-ald, and HA-ADH was 1:1:2, and the solid content of the hydrogel formed after HA-TP and β-CD-ald were dissolved in PBS was not less than 1%.

[0067] The gelation schematic diagram and injectability demonstration are as Figure 2As shown in A and 2B, the hydrogel was subjected to rheological tests, and the results are as Figure 2 shown in C and 2D, demonstrating its good shear thinning and recoverability.

[0068] In the SHG hydrogel, TP grafted on the HA molecular chain is the guest, and the cyclodextrin of β-CD-ald is the host. Through host-guest interaction, β-CD-ald is complexed onto the HA molecular chain, and then the hydrazide group of HA-ADH and the aldehyde group of β-CD-ald form a Schiff base interaction to form a hydrogel network of HA molecular chains. To confirm the formation of the above host-guest interaction and Schiff base interaction, 1 1H nuclear magnetic resonance and FT-IR tests were carried out, and the results are as Figure 3 shown. From the FT-IR spectrum ( Figure 3 a), it can be seen that the SHG hydrogel shows a characteristic absorption peak of C=N, which is in line with the Schiff base structure, proving that the SHG hydrogel is formed by Schiff base interaction. From the 1 1H nuclear magnetic resonance spectrum ( Figure 3 b), it can be seen that the hydrogen on the benzene ring of TP shifts to a higher field, confirming the formation of the host-guest interaction.

[0069] Preparation of the comparative example:

[0070] 1. Preparation steps of acrylated β-cyclodextrin: Dissolve β-cyclodextrin at 6% (w / v) in dimethylformamide (DMF), add 4.5% (v / v) triethylamine, and lower the temperature of the system to 0 °C; dropwise add acryloyl chloride in an amount 7 times the molar amount of cyclodextrin to the system, stir for 12 hours, filter off triethylamine hydrochloride, and obtain a clear solution; concentrate the solution and then drop it into acetone to obtain a white precipitate, wash the obtained white precipitate with acetone, and finally dry it under vacuum to obtain acrylated β-cyclodextrin (Ac-β-CD).

[0071] Dissolve the tert-butylphenylacetic acid esterified hyaluronic acid and acrylated β-cyclodextrin prepared according to the method of the above example in PBS solution, add photoinitiator I 2959, and under the condition of ultraviolet light initiation at 365 nm, a dynamic cross-linked hydrogel HG formed based on host-guest interaction is obtained.

[0072] 2. Prepare aldehyde-functionalized hyaluronic acid by reacting sodium periodate with hyaluronic acid. Dissolve hyaluronic acid in deionized water, add sodium periodate after complete dissolution, react at room temperature for 2 hours, add ethylene glycol and react for 1 hour, dialyze the reaction solution in deionized water for 3 days, and freeze-dry to obtain aldehyde-functionalized hyaluronic acid. Dissolve HA-ADH and aldehyde-functionalized hyaluronic acid prepared according to the above method in PBS solution to obtain a hydrogel S based on Schiff base.

[0073] Example 2

[0074] The SHG hydrogel of Example 1, the control hydrogels HG and S were subjected to creep tests. The method was to first apply a constant force to the hydrogel sample for 1 min while recording the change in strain over time. Then the constant force was removed while recording the change in strain over time, and the sample recovered for 1800 s after the force was removed. The experimental results are as Figure 4 shown in a. The experimental results show that the SHG hydrogel has good plasticity, showing nearly 40% plasticity (as Figure 4 shown in b). It has been proven in the literature that hydrogels with excellent plasticity can promote cell migration and endothelial cell angiogenesis, and hydrogels with a high degree of plasticity enable better spheroid formation of cancer cells. The hydrogel of the present invention improves the plasticity of the hydrogel through tandem dynamic bonds. The plasticity degree of the tandem dynamic crosslinked hydrogel is close to 40%. Compared with the control example, the plasticity degree of the hydrogel HG formed based on host-guest interaction is only about 30%, and the plasticity degree of the hydrogel S formed by pure Schiff base action is only about 25%.

[0075] Example 3

[0076] The SHG hydrogel prepared in Example 1 was used for the culture of 143 - B cells. The 143 - B cells in the culture dish were digested with trypsin, centrifuged, counted, and an appropriate number of cells were resuspended in the B solution of the SHG hydrogel to make the cells evenly distributed in the B solution. Then, 25 μL of the A solution and the B solution containing cells were mixed evenly and added to the mold, and left standing for 30 min to gel the solution to obtain a 50 μL SHG hydrogel containing 20W 143 - B cells. Then it was placed in a 24 - well plate for culture, and 1 mL of DMEM medium (Gibco) was added to each well, and the medium was changed every two days. The same number of 143 - B cells were encapsulated in the control hydrogels HG and S under the same culture conditions for culture. By comparing the cell culture effects of the control hydrogels HG and S under the same conditions, it was found that after culturing single cells in the SHG hydrogel for 7 days, cell spheres with a diameter of nearly greater than 60 μm could be cultured. After culturing in the hydrogel (HG) crosslinked by pure host - guest interaction for 7 days, the cell spheres were only about 40 μm, and after culturing in the pure Schiff base crosslinked hydrogel (S) for 7 days, the diameter of the cell spheres was about 30 μm (as Figure 5 shown).

[0077] Example 4

[0078] Using the SHG hydrogel prepared in Example 1, the culture of HepG2 cells was carried out. The human-derived HepG2 in the culture dish was digested with trypsin, centrifuged, counted, and an appropriate number of cells were resuspended with Solution B of the SHG hydrogel to make the cells evenly distributed in Solution B. Then, 25 μL of Solution A and the Solution B containing cells were mixed evenly and added into a mold, and left standing for 30 min to gel the solution to obtain a 50-μL SHG hydrogel containing 20W HepG2. Then, it was placed in a 24-well plate for culture, and 1 mL of DMEM medium (Gibco) was added to each well, and the medium was changed every two days. The same number of HepG2 cells were encapsulated in the HG hydrogel and S hydrogel of the comparative example and cultured under the same culture conditions.

[0079] After degradation on the seventh day, the cells were re-encapsulated into the hydrogel. After culturing for another 7 days, the cell spheres cultured in the SHG hydrogel could grow to 100 μm, and the cell compatibility between the cells and the material was good (as Figure 6 shown). The live / dead staining of HepG2 cells after culturing in the hydrogel for 7 days (7D 10X) was as Figure 7 shown. On the 7th day, the SHG hydrogel was degraded with glycine or adamantane hydrochloride, which could effectively separate the cells from the hydrogel and perform secondary encapsulation. The results of immunofluorescence staining showed (see Figure 8 ) that HepG2 cells expressed stemness genes including NANOG and SOX2 within 7 days, but the expression on the 14th day was much greater than that on the 7th day.

[0080] Example 5

[0081] In this example, further, HA-TP was reacted with methacrylic anhydride to prepare methacrylic HA-TP, and then the bioactive thiol-terminated RGD (GCGYGRGDSPG) peptide was covalently coupled to the methacrylic HA-TP chain through Michael addition reaction. Then, β-mercaptoethanol was added to react with the remaining methacrylate to obtain HA-TP-RGD. GCGYGRGDSPG was modified on HA-TP (substitution degree Ds = 3%, H 1 The nuclear magnetic resonance spectrum was as Figure 9As shown, it is used to prepare Solution A for the three-dimensional culture of MSC cells. The rBMSC cells in the culture dish are digested with trypsin, centrifuged, counted, and an appropriate number of cells are resuspended in Solution B of the SHG hydrogel to evenly distribute the cells in Solution B. Then, 25 μL of Solution A and the Solution B containing cells are mixed evenly and added to the mold, and left standing for 30 min to gel the solution to obtain a 50 μL SHG hydrogel containing 50W rBMSC cells. Then it is placed in a 24-well plate for culture, and 1 mL of α-MEM medium (Gibco) is added to each well, and the medium is changed every two days. The hydrogel from which the medium is removed is washed with PBS and then fixed with 4% paraformaldehyde for 30 min, and then permeabilized with 0.25% Triton-X100 for 30 min. After permeabilization, the sample is incubated with a PBS solution containing 3% bovine serum albumin for 2 h, then the sample is incubated with α-actin, and finally the nucleus is stained with DAPI (1:1000). After all the staining operations are completed, fluorescence imaging is performed with a Zeiss confocal microscope (Zeiss).

[0082] The light microscope image (10X) of culturing MSC with the SHG hydrogel is as Figure 10 shown in a. The live / dead staining of MSC after culturing in the hydrogel (10D 10X) and the immunofluorescence staining picture of MSC (7D 20X) are respectively as Figure 10 shown in b and 10c. From the light microscope image of the cells, the live / dead staining, and the immunofluorescence staining results, it shows that the hydrogel can promote the spreading of MSC cells and serves as a material for regulating the life of stem cells.

[0083] Example 6

[0084] In this example, the transwell cell migration experiment is used to investigate the cell migration in the SHG hydrogel. The volume of each hydrogel is 30 mL. Then, 100 mL of hMSC (2×10 6 / ml) culture medium is added to the top of the hydrogel, and 770 mL of growth medium containing 10 ng / mL SDF-1 is added to the 24-well plate. After incubation for 2 h, the hydrogel is fixed with 4% paraformaldehyde and stained with DAPI. The distribution of cells in the hydrogel is observed with a confocal microscope. The experimental results show that the migration distance of cells in the SHG hydrogel is significantly greater than that in the HG hydrogel and the S hydrogel.

Claims

1. A dynamic crosslinked supramolecular hydrogel is formed by self-assembly of a biomacromolecule modified with a hydrophobic group, a cyclodextrin modified with an active carbonyl group, and a biomacromolecule modified with an amino or hydrazide group, wherein the biomacromolecule is selected from hyaluronic acid, gelatin, chitosan, dextran, pectin, and carboxymethyl cellulose.

2. The dynamic crosslinked supramolecular hydrogel according to claim 1, wherein The hydrophobic group is selected from adamantane, tert-butylbenzene, acetylsalicylic acid, ibuprofen, menthol, geraniol, and cholic acid.

3. The dynamic crosslinked supramolecular hydrogel according to claim 1 or 2, wherein the active carbonyl in the cyclodextrin modified with an active carbonyl group is an aldehyde group; the amino group is hexamethylenediamine, and the hydrazide group is adipic dihydrazide or succinic dihydrazide.

4. A method for preparing a dynamic crosslinked supramolecular hydrogel, comprising the following steps: (1) Prepare a biomacromolecule modified with a hydrophobic group, a cyclodextrin modified with an active carbonyl group, and a biomacromolecule modified with an amino or hydrazide group respectively; wherein the biomacromolecule is selected from hyaluronic acid, gelatin, chitosan, dextran, pectin, and carboxymethyl cellulose; (2) Mix the biomacromolecule modified with a hydrophobic group and the cyclodextrin modified with an active carbonyl group to prepare aqueous solution A; prepare aqueous solution B from the biomacromolecule modified with an amino or hydrazide group; (3) Mix aqueous solutions A and B to obtain a hydrogel.

5. The preparation method according to claim 4, characterized in that, The hydrophobic group is selected from adamantane, tert-butylbenzene, acetylsalicylic acid, ibuprofen, menthol, geraniol, and cholic acid.

6. The preparation method according to claim 4, characterized in that The biomacromolecule modified with a hydrophobic group is tert-butylphenylacetic acid esterified hyaluronic acid, the cyclodextrin modified with an active carbonyl group is aldehyde group modified cyclodextrin, and the biomacromolecule modified with an amino or hydrazide group is adipic dihydrazide modified hyaluronic acid.

7. The preparation method according to claim 6, characterized in that, After mixing aqueous solutions A and B, the mass-volume concentration ratio of tert-butylphenylacetic acid esterified hyaluronic acid, aldehyde group modified cyclodextrin, and adipic dihydrazide modified hyaluronic acid is 1:(0.8 - 1.2):(1.5 - 2.5).

8. A method for culturing cells in vitro using the dynamic cross-linked supramolecular hydrogel according to claim 1 or the dynamic cross-linked supramolecular hydrogel obtained by the preparation method according to claim 4, characterized in that, Comprising the following steps: Mix the biomacromolecule modified with a hydrophobic group and the cyclodextrin modified with an active carbonyl group to prepare aqueous solution A1; dissolve the biomacromolecule modified with an amino or hydrazide group in water or a buffer solution, add cells and mix evenly to prepare aqueous solution B1; mix aqueous solutions A1 and B1, and after obtaining a hydrogel, add a cell culture medium for three-dimensional culture.

9. The application of the dynamic crosslinked supramolecular hydrogel according to any one of claims 1 - 3 in three-dimensional cell culture, drug delivery, preparation of injectable medicaments, and tissue engineering.

10. The application of the dynamic crosslinked supramolecular hydrogel obtained by the preparation method according to any one of claims 4 - 7 in three-dimensional cell culture, drug delivery, preparation of injectable medicaments, and tissue engineering.

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