A hydrogel with a double cross-linked network, its preparation method and application

Abstract

The present invention belongs to the technical field of hydrogel materials, and particularly relates to a double-crosslinked network hydrogel and its preparation method and application. The raw materials for preparing the double-crosslinked network hydrogel of the present invention include methacryloyl-based photosensitive materials, chondroitin sulfate salts, drugs, and photoinitiators; the methacryloyl-based photosensitive materials do not include methacryloyl hyaluronic acid. In the present invention, crosslinking occurs between the methacryloyl-based photosensitive materials and the chondroitin sulfate salts, and then the drugs are further crosslinked to obtain a double-crosslinked network hydrogel. There are binding sites among the three, and the pore size of the crosslinked double-crosslinked network hydrogel is larger, so that the hydrogel can continuously and slowly release drugs, has good mechanical properties, has a good effect of promoting cell proliferation, and has good stability in neutral and acidic environments.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel materials, and particularly relates to a double-crosslinked network hydrogel and its preparation method and application. Background Art

[0002] Increasing physical activity has been shown to have a positive effect on preventing and improving various diseases, including brain diseases such as Alzheimer's disease, cancer, diabetes, cardiovascular diseases, etc. In addition, it also has a certain effect on the rehabilitation of fractures. However, the movement of fracture patients will cause micro-movement of the fracture ends, affecting the fracture rehabilitation. Exercise mimetics are an emerging treatment method, and the development of exercise mimetics has great potential in preventing and treating diseases. In order to develop new exercise mimetics, it is crucial to understand the mechanisms mediating the therapeutic effects of physical activity at the molecular, cellular, and systemic levels. Different molecules and signaling pathways are associated with the enhancement of adult neural and synaptic activities after exercise. These molecules include brain-derived neurotrophic factor (BDNF), tropomyosin-related kinase B (TrkB), and related neurotrophic factor receptor signaling pathways, etc. For bones, related experiments have shown that BDNF can promote the differentiation and osteogenesis of bone mesenchymal stem cells and promote the fracture healing process. However, the application of BDNF as a drug in clinical trials is not ideal, probably due to its relatively short half-life and poor delivery characteristics. In addition, there are currently studies on pH-sensitive drug delivery carriers, but their carriers decompose rapidly in an acidic environment and there is a possibility of decomposition in the inflammatory state of the body, unable to achieve slow release, and thus unable to achieve the purpose of prolonging the effective therapeutic effect.

[0003] Therefore, it is of great significance to provide a hydrogel that can sustainably and slowly release drugs, has good mechanical properties, and has a good promoting effect on cell proliferation. Summary of the Invention

[0004] The present invention aims to solve one or more of the above-mentioned technical problems existing in the prior art, and at least provides a beneficial choice or creation condition. Specifically, the present invention provides a double-crosslinked network hydrogel, which can sustainably and slowly release drugs, has good mechanical properties, and has a good promoting effect on cell proliferation.

[0005] The inventive concept of the present invention: The double-crosslinked network hydrogel of the present invention comprises a methacryloyl-based photosensitive material, chondroitin sulfate salt, a drug, and a photoinitiator; the methacryloyl-based photosensitive material does not include methacryloyl hyaluronic acid. Under the action of the photoinitiator, the methacryloyl-based photosensitive material and the chondroitin sulfate salt are crosslinked, and then the drug is further crosslinked, so that there are binding sites among the three, and the pore size of the crosslinked double-crosslinked network hydrogel is larger, thereby enabling the hydrogel to sustainably and slowly release drugs, have good mechanical properties, and have a good promoting effect on cell proliferation.

[0006] Therefore, the first aspect of the present invention provides a hydrogel with a double cross - linked network.

[0007] Specifically, the hydrogel with a double cross - linked network includes a methacryloyl - type photosensitive material, chondroitin sulfate salt, a drug, and a photoinitiator;

[0008] The methacryloyl - type photosensitive material does not include methacryloyl hyaluronic acid.

[0009] Preferably, the methacryloyl - type photosensitive material includes methacryloyl gelatin.

[0010] Preferably, the raw materials for preparing the hydrogel further include a solvent.

[0011] Preferably, the solvent includes water.

[0012] Preferably, the chondroitin sulfate salt includes strontium chondroitin sulfate.

[0013] Preferably, the drug includes 7,8 - dihydroxyflavone.

[0014] Specifically, 7,8 - dihydroxyflavone (7,8 - DHF) is a flavonoid derived from plants, a functional BDNF mimetic, which can overcome the defects of the relatively short half - life and poor delivery characteristics of BDNF as a drug, and can activate the TrkB - related signaling pathway. However, as a lipophilic drug, the physical property of poor water solubility of 7,8 - DHF limits its clinical application. Therefore, in the present invention, 7,8 - dihydroxyflavone acts together with the methacryloyl - type photosensitive material and chondroitin sulfate salt to obtain a hydrogel with light responsiveness and a double cross - linked network. There are binding sites among the three, and 7,8 - dihydroxyflavone is loaded on the hydrogel. The hydrogel is stable in neutral and acidic environments, can continuously and slowly release 7,8 - dihydroxyflavone, and has good mechanical properties.

[0015] Specifically, the combination of specific types of methacryloyl - type photosensitive materials, chondroitin sulfate salts, and drugs can better realize the performance of the hydrogel, making the hydrogel have better stability, slow - release performance, and mechanical properties.

[0016] Preferably, the photoinitiator includes lithium phenyl(2,4,6 - trimethylbenzoyl)phosphate (LAP).

[0017] The principle of the LAP photoinitiator is based on the reaction of photo - acid generation. When the LAP photoinitiator is exposed to ultraviolet or blue light, its molecules will be excited to a high - energy state. In this high - energy state, the LAP photoinitiator molecules can generate an acidic proton (H +) The acidic protons can further react and initiate a photochemical reaction, resulting in the growth and crosslinking of molecular chains.

[0018] Preferably, the mass ratio of the methacryloyl-based photosensitive material to the chondroitin sulfate salt is 5:(0.5 - 1.2); more preferably, the mass ratio of the methacryloyl-based photosensitive material to the chondroitin sulfate salt is 5:(0.8 - 1.1); even more preferably, the mass ratio of the methacryloyl-based photosensitive material to the chondroitin sulfate salt is 5:1.

[0019] Preferably, the mass ratio of the methacryloyl-based photosensitive material to the drug is 5:(0.008 - 0.012); more preferably, the mass ratio of the methacryloyl-based photosensitive material, chondroitin sulfate salt, and drug is 5:(0.009 - 0.011); even more preferably, the mass ratio of the methacryloyl-based photosensitive material, chondroitin sulfate salt, and drug is 5:0.01.

[0020] Specifically, the methacryloyl-based photosensitive material, chondroitin sulfate salt, and drug need to satisfy the above proportional relationship. For example, if the mass proportion of the chondroitin sulfate salt is too large, it will increase the strength of the hydrogel, correspondingly reduce the toughness of the hydrogel, and thus cannot well simulate human soft tissues.

[0021] Preferably, the mass ratio of the methacryloyl-based photosensitive material to the photoinitiator is (16 - 25):1; more preferably, the mass ratio of the methacryloyl-based photosensitive material to the photoinitiator is (18 - 22):1; even more preferably, the mass ratio of the methacryloyl-based photosensitive material to the photoinitiator is 20:1.

[0022] The second aspect of the present invention provides a preparation method of the hydrogel with a double cross-linked network as described in the first aspect of the present invention.

[0023] Specifically, the preparation method of the hydrogel with a double cross-linked network includes the following steps:

[0024] Mix the raw material components, heat, and irradiate with light to obtain the hydrogel.

[0025] Preferably, the preparation method of the hydrogel with a double cross-linked network includes the following steps:

[0026] (1) Mix the methacryloyl-based photosensitive material, photoinitiator, and solvent, and heat to obtain a first solution;

[0027] (2) Mix the chondroitin sulfate salt and the first solution obtained in step (1), and heat to obtain a second solution;

[0028] (3) Mix the drug with the second solution obtained in step (2), and heat to obtain a third solution;

[0029] (4) Irradiate the third solution obtained in step (3) with light to prepare the hydrogel.

[0030] Preferably, in steps (1), (2), and (3), the heating temperature is 50 - 70 °C; more preferably, in steps (1), (2), and (3), the heating temperature is 55 - 65 °C; even more preferably, in steps (1), (2), and (3), the heating temperature is 60 °C.

[0031] Preferably, step (1) is specifically as follows: First, mix the photoinitiator with the solvent, heat, and vortex - oscillate, then add the methacryloyl - type photosensitive material, heat, and vortex - oscillate to obtain the first solution.

[0032] Preferably, in step (1), the time of vortex - oscillation is 4 - 6 min; more preferably, in step (1), the time of vortex - oscillation is 4.5 - 5.5 min; even more preferably, in step (1), the time of vortex - oscillation is 5 min.

[0033] Preferably, in step (1), the solvent includes water.

[0034] Preferably, the dosage ratio of the photoinitiator to the solvent is 2.5 g:(0.8 - 1.2) mL; more preferably, the dosage ratio of the photoinitiator to the solvent is 2.5 g:(0.9 - 1.1) mL; even more preferably, the dosage ratio of the photoinitiator to the solvent is 2.5 g:1 mL.

[0035] Preferably, in step (2), the concentration of chondroitin sulfate salt in the second solution is 0.5 - 1.2% (w / v); more preferably, in step (2), the concentration of chondroitin sulfate salt in the second solution is 0.8 - 1.1% (w / v); even more preferably, in step (2), the concentration of chondroitin sulfate salt in the second solution is 1% (w / v).

[0036] Preferably, in step (2), vortex - oscillate after heating to the corresponding temperature.

[0037] Preferably, in step (2), the time of vortex - oscillation is 8 - 12 min; more preferably, in step (2), the time of vortex - oscillation is 9 - 11 min; even more preferably, in step (2), the time of vortex - oscillation is 10 min.

[0038] Specifically, in step (2), a drug carrier is obtained after crosslinking of a methacryloyl photosensitive material and chondroitin sulfate. This carrier is in a liquid state and is light-responsive. The liquid state facilitates transportation and injection, and after being injected into a predetermined position, it can be quickly stimulated to crosslink into a gel and play a role.

[0039] Preferably, in step (3), first mix the drug and the solvent to obtain a drug solution, and then add the drug solution to the second solution obtained in step (2), heat it, and vortex-oscillate it to obtain a third solution.

[0040] Preferably, the dosage ratio of the drug to the solvent is (0.8 - 1.2) mg : 100 μL; more preferably, the dosage ratio of the drug to the solvent is (0.9 - 1.1) mg : 100 μL; even more preferably, the dosage ratio of the drug to the solvent is 1 mg : 100 μL.

[0041] Preferably, the solvent includes dimethyl sulfoxide.

[0042] Preferably, the volume ratio of the drug solution to the second solution is 1 : (80 - 120); more preferably, the volume ratio of the drug solution to the second solution is 1 : (90 - 110); even more preferably, the volume ratio of the drug solution to the second solution is 1 : 100.

[0043] Specifically, when the drug is 7,8-DHF, the solvent for 7,8-DHF is dimethyl sulfoxide (DMSO). DMSO is commonly used for cell cryopreservation and has certain toxicity. At the same time, DMSO is also a commonly used organic solvent for dissolving substances that are poorly soluble in water. In this product, the concentration of DMSO is controlled to be less than or equal to 1%, which will not cause negative effects.

[0044] Preferably, in step (3), the time for vortex-oscillation is 8 - 12 min; more preferably, in step (3), the time for vortex-oscillation is 9 - 11 min; even more preferably, in step (3), the time for vortex-oscillation is 10 min.

[0045] Preferably, step (4) is specifically that the third solution is heated in a water bath and irradiated with light to obtain the hydrogel.

[0046] Preferably, the temperature for water bath heating is 30 - 45 °C, and the time for water bath heating is 8 - 12 min; more preferably, the temperature for water bath heating is 33 - 40 °C, and the time for water bath heating is 9 - 11 min; even more preferably, the temperature for water bath heating is 37 °C, and the time for water bath heating is 10 min.

[0047] Specifically, water bath heating ensures that the third solution is in a liquid state.

[0048] Preferably, the irradiation is selected from any one of ultraviolet excitation lamp irradiation and blue light excitation lamp irradiation.

[0049] The third aspect of the present invention provides a gel product.

[0050] Specifically, the gel product includes the hydrogel described in the first aspect of the present invention.

[0051] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows:

[0052] (1) In the present invention, through the crosslinking between methacryloyl-based photosensitive materials and chondroitin sulfate salts, and then further crosslinking of the drug, a hydrogel with a double crosslinked network is obtained. There are binding sites among the three, and the pore size of the crosslinked hydrogel with a double crosslinked network is larger. Furthermore, it can continuously and slowly release the drug, has good mechanical properties, has a good promoting effect on cell proliferation, and the hydrogel has good stability in neutral and acidic environments.

[0053] (2) In the present invention, methacryloyl-based photosensitive materials and chondroitin sulfate salts are crosslinked to obtain a carrier for the drug. This carrier is in a liquid state and is light-responsive. The liquid state is convenient for transportation and injection. After being injected into a predetermined position, it can be quickly excited to crosslink into a gel and play a role.

[0054] (3) The preparation process of the present invention is simple and convenient for industrial mass production. Description of the Drawings

[0055] Figure 1 It is a physical diagram of the hydrogel with a double crosslinked network prepared in Example 1 of the present invention;

[0056] Figure 2 It is an SEM diagram of the GelMA, GS, and GSD solutions prepared in Example 1 of the present invention after gel formation and freeze-drying;

[0057] Figure 3 It is a Fourier infrared spectrum diagram of the GelMA, GS, and GSD solutions prepared in Example 1 of the present invention after gel formation;

[0058] Figure 4 It is an energy spectrum diagram of the hydrogel with a double crosslinked network prepared in Example 1 of the present invention after freeze-drying;

[0059] Figure 5 It is a storage modulus curve diagram of the hydrogels of Example 1 and Comparative Examples 1-5 of the present invention;

[0060] Figure 6 It is a modulus curve diagram of the GelMA, GS, and GSD solutions prepared in Example 1 of the present invention after gel formation;

[0061] Figure 7 Release performance curve of 7,8-DHF in the hydrogels prepared in Example 1 and Comparative Examples 1-5 of the present invention;

[0062] Figure 8 Proliferation effect diagram of the hydrogels prepared in Example 1 and Comparative Examples 1-5 of the present invention on bone marrow mesenchymal stem cells;

[0063] Figure 9 Proliferation effect diagram of the leachates of GelMA, GS, and GSD in Example 1 of the present invention on bone marrow mesenchymal stem cells;

[0064] Figure 10 Live / dead cell staining result diagram of the leachates of GelMA, GS, and GSD in Example 1 of the present invention on bone marrow mesenchymal stem cells;

[0065] Figure 11 Live / dead cell staining result diagram of the leachates of the hydrogels prepared in Example 1 and Comparative Examples 1-5 of the present invention on bone marrow mesenchymal stem cells. Detailed implementation manners

[0066] In order to make those skilled in the art more clearly understand the technical solutions of the present invention, the following examples are listed for illustration. It should be noted that the following examples do not limit the protection scope required by the present invention.

[0067] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial channels or can be obtained by existing known methods.

[0068] Example 1

[0069] A double-crosslinked network hydrogel, the raw materials for its preparation include gelatin methacrylate (GelMA), strontium chondroitin sulfate (SrCS), 7,8-DHF, and LAP photoinitiator; the mass ratio of GelMA, SrCS, and 7,8-DHF is 5:1:0.01; the mass ratio of GelMA and LAP photoinitiator is 20:1.

[0070] A preparation method of a double-crosslinked network hydrogel, comprising the following steps:

[0071] (1) Take 12.5 mg of LAP photoinitiator, add 5 mL of pure water, heat to 60 °C, and vortex for 5 min to obtain a photoinitiator solution; weigh 250 mg of freeze-dried GelMA, add it to the photoinitiator solution, heat to 60 °C, and vortex for 5 min to obtain a GelMA solution before gelation (the concentration of GelMA in the solution is 5% (w / v));

[0072] (2) Take 50 mg of powdered SrCS and add it to the GelMA solution obtained in step (1). Heat the mixture to 60 °C and vortex for 10 min to obtain the GelMA / SrCS solution before gelation, denoted as the GS solution (the concentration of SrCS in the GS solution reaches 1% (w / v));

[0073] (3) Take 5 mg of powdered 7,8-DHF and add it to 500 μL of dimethyl sulfoxide (DMSO) to obtain a 7,8-DHF solution (the concentration of 7,8-DHF is 1% (w / v)). Take 50 μL of the 1% 7,8-DHF solution and add it to 5 mL of the GS solution obtained in step (2). Heat the mixture to 60 °C and vortex for 10 min to obtain the 7,8-DHF@GelMA / SrCS solution, denoted as the GSD solution (the concentration of 7,8-DHF is 0.01% (w / v));

[0074] (4) Put the prepared GSD solution into a brown glass bottle, seal it, keep it away from light, and store it at 4 °C. Before use, heat it in a water bath at 37 °C for 10 min to ensure it melts into a liquid state. Then, draw the GSD solution into a syringe and inject it into the application site. Irradiate it with an ultraviolet excitation lamp for 30 s to form a gel, obtaining a hydrogel with a double-crosslinked network.

[0075] The physical picture of the hydrogel prepared in Example 1 is as Figure 1 shown, where Figure 1 Figure (a) is the picture before gelation, Figure 1 and Figures (b) and (c) are the pictures after gelation. It can be Figure 1 seen that before irradiation with the ultraviolet excitation lamp, GSD is in a liquid state and can flow; after gelation by ultraviolet irradiation, it is in a gel state and does not have fluidity.

[0076] Comparative Example 1

[0077] The difference between Comparative Example 1 and Example 1 is only that in Comparative Example 1, an equal amount of hyaluronic acid methacrylate (HAMA) is used to replace GelMA in Example 1, and the others are the same as in Example 1.

[0078] Comparative Example 2

[0079] The difference between Comparative Example 2 and Example 1 is only that in Comparative Example 2, an equal amount of chondroitin sulfate CS is used to replace SrCS in Example 1, and the others are the same as in Example 1.

[0080] Comparative Example 3

[0081] The difference between Comparative Example 3 and Example 1 is only that in step (2) of Comparative Example 3, the addition amount of SrCS is 100 g, so that the concentration of SrCS in the GS solution reaches 2% (w / v), and the others are the same as in Example 1.

[0082] Comparative Example 4

[0083] The difference between Comparative Example 4 and Example 1 is only that in Comparative Example 4, an equal amount of methacryloyl hyaluronic acid (HAMA) is used to replace GelMA in Example 1, and an equal amount of chondroitin sulfate CS is used to replace SrCS in Example 1, and the others are the same as in Example 1.

[0084] Comparative Example 5

[0085] The difference between Comparative Example 5 and Example 1 is only that in Comparative Example 5, an equal amount of methacryloyl hyaluronic acid (HAMA) is used to replace GelMA in Example 1, and the addition amount of SrCS in step (2) of Comparative Example 5 is 100 g, so that the concentration of SrCS in the GS solution reaches 2% (w / v), and the others are the same as in Example 1.

[0086] Performance Test

[0087] 1. SEM Observation

[0088] The GelMA solution, GS solution, and GSD solution prepared in Example 1 were irradiated with a 405 nm wavelength ultraviolet light source for 30 s to form a gel. The prepared hydrogel was pre-cooled in a -80 °C refrigerator for 30 min in advance, and then quickly placed in a vacuum freeze dryer, started, and dried for 48 h to obtain the freeze-dried hydrogel. The SEM images of the freeze-dried hydrogel are as Figure 2 shown. Among them, Figure 2 Figure (a) in it is the SEM image of the GelMA solution after gel formation and freeze-drying, Figure 2 Figure (b) in it is the SEM image of the GS solution after gel formation and freeze-drying, Figure 2 Figure (c) in it is the SEM image of the GSD solution after gel formation and freeze-drying.

[0089] It can be seen from Figure 2 that in the gel obtained by adding SrCS to GelMA, the pore size does not increase significantly, and its mechanical properties are not affected; while after the GSD solution obtained by incorporating 7,8-DHF into GS is gelated and freeze-dried, its pore size increases significantly, which proves that 7,8-DHF has cross-linked with GS on the side, and the larger pore size represents better biocompatibility and more excellent drug release performance.

[0090] 2. Fourier Transform Infrared Spectroscopy (FTIR) Test

[0091] The FTIR images of the GelMA solution, GS solution, and GSD solution prepared in Example 1 after gel formation (the gel formation method is the same as that in the SEM observation) are as Figure 3 shown. Among them, Figure 3 Figure (a) in it is the comparative FTIR image of GelMA adding SrCS to form GS,Figure 3 In Figure (b), it is the FTIR image comparison diagram of GS adding 7,8-DHF to form GSD. The abscissa Wavenumbers (cm -1 ) both represent the wave number, and the ordinate Transmittance both represents the transmittance.

[0092] From Figure 3 it can be seen that the characteristic peaks of GelMA and GS have shifted, indicating the formation of new bonds, which shows that there are binding sites among GelMA, SrCS, and 7,8-DHF.

[0093] 3. Energy Dispersive X-ray Spectrometer (EDS) Analysis

[0094] The hydrogel of Example 1 was irradiated by an ultraviolet laser lamp to form a gel, then washed, and then freeze-dried to obtain the freeze-dried hydrogel. Among them, the washing method was soaking and washing with distilled water. Specifically, the hydrogel was placed in distilled water to fully swell it, and then the distilled water was changed after soaking for a certain time. This process was repeated more than 3 times to remove the water-soluble impurities in the gel.

[0095] The hydrogel prepared in Example 1 after freeze-drying was tested by an Energy Dispersive X-ray Spectrometer (EDS), and the energy spectrum curve is as Figure 4 shown.

[0096] From Figure 4 it can be seen that C, N, O, and Sr exist in the hydrogel; among them, the weight percentages of C, N, O, and Sr elements are 54.75%, 11.92%, 27.87%, and 5.45% respectively; C, N, and O come from GelMA and 7,8-DHF, and Sr comes from SrCS. The Sr element still exists in the washed hydrogel, indicating that SrCS binds to GelMA and is not easily eluted.

[0097] 4. Storage Modulus Test

[0098] The storage modulus of the hydrogels prepared in Example 1 and Comparative Examples 1-5 was tested by a rotational rheometer, and the storage modulus curve is as Figure 5 shown, where the abscissa time represents time and the ordinate G’ represents the storage modulus.

[0099] From Figure 5It can be seen that the hydrogel of Example 1 has a lower storage modulus, indicating that it exhibits higher toughness rather than hydrogel strength and can better simulate human soft tissues. The replacement of HAMA, the replacement of CS, and the increase in the SrCS concentration in Comparative Examples 1-5 all enhance the hydrogel strength and correspondingly reduce the toughness of the hydrogel, making it unable to well simulate human soft tissues. This may be because HAMA has a higher storage modulus compared to GelMA, SrCS has a lower storage modulus relative to the HAMA group, and the binding of strontium ions to CS reduces the binding sites between CS and GelMA, resulting in a low storage modulus of the hydrogel when SrCS is used in Example 1. The higher storage modulus of the high-concentration SrCS in Comparative Example 3 is due to the fact that the binding sites between GelMA and SrCS have not reached the upper limit.

[0100] In addition, a rotational rheometer was used to test the modulus of the hydrogels formed from the GelMA, GS, and GSD solutions prepared in Example 1. The modulus curves are as Figure 6 shown. Among them, the abscissa Time(s) represents time (seconds), and the ordinate Modulus(Pa) represents modulus (Pascals). GelMA-G’ represents the storage modulus of GelMA, GS-G’ represents the storage modulus of GS, GSD-G’ represents the storage modulus of GSD, GelMA-G” represents the loss modulus of GelMA, GS-G” represents the loss modulus of GS, and GSD-G” represents the loss modulus of GSD.

[0101] From Figure 6 it can be seen that the storage modulus and loss modulus of GS are greater than those of GelMA, indicating that the structure and strength of GS are enhanced after incorporating SrCS; the modulus of GSD is significantly lower than that of GS, indicating that the structure of GSD has changed after loading 7,8-DHF, and the chemical bonds therein are bound by 7,8-DHF, resulting in a decrease in modulus.

[0102] 5. Release performance test

[0103] The release performance of 7,8-DHF in the hydrogels prepared in Example 1 and Comparative Examples 1-5 was tested, that is, the cumulative release amount of 7,8-DHF was analyzed and calculated. The specific method is as follows:

[0104] By collecting 7,8-DHF solutions with different concentration gradients and constructing their standard concentration curves. Specifically, prepare 7,8-DHF aqueous solutions (containing 1% DMSO) with concentrations of 0.5, 1, 2, 5, and 10 μg / mL respectively. Measure them using an ultraviolet spectrophotometer to obtain the corresponding waveform diagrams. Refer to the literature and perform peak analysis to determine that 280 nm is the characteristic peak wavelength of 7,8-DHF. Obtain the values corresponding to each concentration and plot a graph, and perform regression analysis to obtain the formula for the concentration corresponding to the value at 280 nm, thus obtaining the standard concentration curve of the 7,8-DHF solution. Thereafter, by measuring the value of the solution at 280 nm using an ultraviolet spectrophotometer, the corresponding concentration can be calculated, and then the total cumulative release amount can be calculated. Oscillate at a speed of 100 rpm at 37°C, and observe the drug release of the hydrogels in each scheme in 50 mL of phosphate buffer solution (PBS) with pH = 7.4. At the set time points, take out 2 mL of PBS containing 7,8-DHF for analysis, and supplement the same volume of fresh PBS after each sampling.

[0105] The release performance curves of 7,8-DHF in the hydrogels prepared in Example 1 and Comparative Examples 1-5 are as Figure 7 shown, where the abscissa Time (Day) represents time (days), and the ordinate 7,8-DHF release (%) represents the cumulative release amount of 7,8-DHF.

[0106] From Figure 7 it can be seen that the cumulative release amount of 7,8-DHF in the hydrogel of Example 1 is greater than that in the hydrogels of Comparative Examples 1-5. The release of 7,8-DHF in the hydrogel of Example 1 is the fastest. A faster drug release rate indicates a faster action time and a faster dynamic regulation ability. In Comparative Examples 1-5, due to the relatively large structural strength of the hydrogel, the drug release is slowed down. In addition, from the release performance curve of the hydrogel of Example 1, it can be seen that 7,8-DHF is continuously released within 3 days. After the release reaches 40%, the release concentration reaches the peak, and the internal and external concentrations reach equilibrium.

[0107] 6. Cell proliferation test

[0108] The cell viability detection (CCK-8) was used to test the effects of the hydrogels of Example 1 and Comparative Examples 1-5 on the proliferation of bone marrow mesenchymal stem cells (BMSCs). The specific test method is as follows:

[0109] BMSC cells were implanted into a 96-well plate at a density of 1000 per well. The BMSC cells were cultured in the hydrogel extract of Example 1 and Comparative Examples 1-5 with a 7,8-DHF concentration of 0.2 μg / mL for 1 day, 3 days, and 5 days. At these specified time points, 10 μL of CCK-8 reagent and 100 μL of fresh basal α-MEM culture medium were added to each well. After incubating the cells for 2 hours, the absorbance of the cells at a wavelength of 450 nm was tested using an enzyme-linked immunosorbent assay (BioTek, USA) at 37°C. Among them, the preparation method of the hydrogel extract of Example 1 and Comparative Examples 1-5 is as follows: 100 μL of the pre-gel solution is taken respectively, irradiated with ultraviolet light for 30 seconds to form a gel, pre-cooled in a -80°C refrigerator for 30 minutes, and then quickly placed in a vacuum freeze dryer for freeze drying for 48 hours, and then placed in an α-MEM complete medium containing 1% double antibody and 10% fetal bovine serum, and shaken at 37°C and 100 rpm for 24 hours, and then filtered using a 0.22 μm filter membrane to obtain a hydrogel extract with a 7,8-DHF concentration of 0.2 μg / mL.

[0110] Example 1, Comparative Examples 1-5 The proliferation effects of hydrogels on bone marrow mesenchymal stem cells (BMSC) are as follows Figure 8 As shown, the horizontal axes Day1, Day2, and Day3 represent the 1st day, the 3rd day, and the 5th day, respectively, and the bar graphs on the 1st day, the 3rd day, and the 5th day are Example 1, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, and Comparative Example 5 from left to right, and the vertical axis Cell Viability (% of control) represents cell activity.

[0111] Depend on Figure 8 It can be seen that the proliferation-promoting effect of Example 1 on BMSC cells is significantly higher than that of Comparative Examples 1-5, indicating that the hydrogel of the present invention has a good proliferation-promoting effect on BMSC cells.

[0112] The proliferation effect of the extracts of GelMA, GS and GSD in Example 1 on BMSCs was tested, and the specific test method is as follows:

[0113] BMSC cells were seeded into a 96-well plate at a density of 1000 cells per well and cultured in GelMA, GS, and GSD leachates with the same formulation and component content for 1 day, 3 days, and 5 days. At these specified time points, 10 μL of CCK-8 reagent and 100 μL of fresh medium were added to each well. After incubating the cells for 2 hours, the absorbance of the cells at a wavelength of 450 nm was measured using an enzyme-linked immunosorbent assay instrument (BioTek, USA) at 37°C. The preparation method of GelMA, GS, and GSD leachates was as follows: 100 μL of the pre-gel solution was taken, gelled under ultraviolet light for 30 s, pre-cooled in an -80°C refrigerator for 30 min, then quickly placed in a vacuum freeze dryer and freeze-dried for 48 h, and then placed in α-MEM complete medium containing 1% double antibody and 10% fetal bovine serum, shaken at 37°C and 100 rpm for 24 h, and then filtered using a 0.22 μm filter membrane to obtain the leachates of GelMA, GS, and GSD.

[0114] The proliferation effects of the GelMA, GS, and GSD leachates in Example 1 on BMSC are as Figure 9 shown. Among them, the abscissa Day1, Day2, and Day3 represent the 1st day, 3rd day, and 5th day respectively, and the bar graphs on the 1st day, 3rd day, and 5th day are NC, GelMA, Gel-SrCS, and Gel-SrCS@7,8-DHF from left to right in sequence; the ordinate Cell Viability (%of control) represents cell viability; NC represents the blank control group, Gel-SrCS represents GS, 7,8-DHF@GelMA / SrCS represents GSD, ns indicates no statistical difference, * indicates a statistical difference from the NC group, # indicates a statistical difference from the GelMA group, and & indicates a statistical difference from the GS group.

[0115] It can be seen from Figure 9 this that the GelMA group has no obvious biological activity. After adding SrCS, the cell proliferation in the GS group is obvious, indicating that SrCS has a certain promoting effect on proliferation. After adding 7,8-DHF, the cell proliferation in the GSD group is more obvious than that in the GS group, indicating that 7,8-DHF has an obvious promoting effect on the proliferation of BMSC cells.

[0116] 7. Viability staining test of cells

[0117] The leachates of GelMA, GS, and GSD in Example 1 were tested for live / dead cell staining of BMSC cells. The specific test method is as follows: BMSC cells were seeded into a 96-well plate at a density of 1000 cells per well. The BMSC cells were cultured in the leachates of GelMA, GS, and GSD with the same component content ratios for 1 day, 3 days, and 5 days. At these specified time points, the staining agent was prepared according to the Live / Dead Cell Staining Kit from Beyotime and added to each well in sequence. After incubating in the dark for 30 min, a confocal microscope was used with a magnification of 10× for photography. Among them, the preparation method of the leachates of GelMA, GS, and GSD was the same as that in the test for the proliferation effect of the leachates of GelMA, GS, and GSD on BMSC cells described above.

[0118] The results of live / dead cell staining of BMSC cells with the leachates of GelMA, GS, and GSD prepared in Example 1 of the present invention are as Figure 10 shown, where D1, D3, and D5 represent day 1, day 3, and day 5, respectively.

[0119] It can be seen from Figure 10 that the live / dead cell staining of BMSC cells corresponds to the results of CCK-8. During the 5-day culture process, the cell density gradually increased with the extension of time. Moreover, the GSD hydrogel had a certain proliferative effect compared with the control groups (GelMA group, GS group), and there were almost no dead cells in all groups, demonstrating that the hydrogel had good biocompatibility, 7,8-DHF was non-toxic to BMSC cells, and had a certain promoting effect on proliferation.

[0120] The results of live / dead cell staining of BMSC cells with the leachates of the hydrogels prepared in Example 1 and Comparative Examples 1-5 of the present invention (day 3) are as Figure 11 shown. Figure 11 Figure (a) shows the results of live / dead cell staining of BMSC cells with the leachate of the hydrogel prepared in Example 1; Figure 11 Figures (b), (c), (d), (e), and (f) respectively show the results of live / dead cell staining of BMSC cells with the leachates of the hydrogels prepared in Comparative Examples 1-5. The preparation method of the leachates of the hydrogels prepared in Example 1 and Comparative Examples 1-5 was the same as that of the hydrogel leachate in the above cell proliferation test.

[0121] It can be seen from Figure 11 that the live / dead cell staining of BMSC cells corresponds to Figure 8 the results of CCK-8. No red fluorescence indicating dead cells appeared in each group. The number of cells in the examples was significantly higher than that in Comparative Examples 1-5, proving that the hydrogel of the present invention had good biocompatibility, and the components of the examples were appropriate and the contents were optimal.

[0122] In summary, in the present invention, crosslinking occurs between a methacryloyl-based photosensitive material and chondroitin sulfate salt, and then the drug is further crosslinked to obtain a hydrogel with a double-crosslinked network. There are binding sites among the three, and the pore size of the crosslinked hydrogel with the double-crosslinked network is larger. As a result, the hydrogel can sustainably and slowly release the drug, has good mechanical properties, has a good effect of promoting cell proliferation, and is stable in neutral and acidic environments.

[0123] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A hydrogel, characterized in that: The raw materials for its preparation include methacryloyl-type photosensitive materials, chondroitin sulfate salt, drugs, and photoinitiators; The methacryloyl photosensitive material is methacryloyl gelatin; the chondroitin sulfate salt is strontium chondroitin sulfate; The drug is 7,8-dihydroxyflavone; The mass ratio of the methacryloyl gelatin to strontium chondroitin sulfate is 5:(0.5-1.2); The mass ratio of the methacryloyl gelatin to 7,8-dihydroxyflavone is 5:(0.008-0.012); The mass ratio of the methacryloyl gelatin to the photoinitiator is (16-25):

1.

2. The hydrogel according to claim 1, characterized in that The raw materials for preparing the hydrogel also include a solvent.

3. The hydrogel according to claim 1, characterized in that The photoinitiator includes lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

4. The method for preparing the hydrogel according to any one of claims 1 to 3, characterized in that: The following steps are involved: The raw material components are mixed, heated and irradiated with light to prepare the hydrogel.

5. The preparation method according to claim 4, characterized in that: The following steps are involved: (1) mixing a methacrylic photosensitive material, a photoinitiator, and a solvent, and heating the mixture to obtain a first solution; (2) mixing the chondroitin sulfate salt and the first solution obtained in step (1), and heating them to obtain a second solution; (3) mixing the drug and the second solution obtained in step (2), and heating them to obtain a third solution; (4) The third solution obtained in step (3) is irradiated with light to obtain the hydrogel.

6. The preparation method according to claim 5, characterized in that: In steps (1), (2) and (3), the heating temperature is 50-70°C.

7. A gel product, characterized in that: The invention comprises the hydrogel according to any one of claims 1 to 3.

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