One-step purified dual-modified hyaluronic acid hydrogel and preparation method thereof

Abstract

The invention relates to a one-step purified dual-modified hyaluronic acid hydrogel and a preparation method thereof, and the preparation method comprises the following steps: under a dark condition, dropwise adding a sodium periodate solution into a sodium hyaluronate solution to carry out hydroformylation reaction, and quenching to obtain a hydroformylated hyaluronic acid reaction solution; cooling the hydroformylated hyaluronic acid reaction solution to 2-6 DEG C, dropwise adding a methacrylic anhydride solution under a dark condition to perform esterification reaction, controlling the pH value of the reaction to be 8-9, and purifying and drying after the reaction is finished to obtain dual modified hyaluronic acid; and dissolving the dual-modified hyaluronic acid and a photoinitiator in a PBS buffer solution to form a precursor solution, and carrying out a photo-crosslinking reaction to obtain the dual-modified hyaluronic acid hydrogel. According to the preparation method disclosed by the invention, aldehydes are firstly subjected to esterification reaction, so that mutual interference is avoided, side reactions are reduced by controlling reaction conditions, and the obtained hydrogel has controllable gelation time, excellent mechanical properties, swelling resistance, long-term stability and good blood compatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a one-step purification dual-modified hyaluronic acid hydrogel and a preparation method thereof. Background Art

[0002] As a major component of the natural extracellular matrix, hyaluronic acid exhibits important application values in the fields of soft tissue repair, drug delivery, and tissue engineering due to its excellent biocompatibility, degradability, and lubricating properties. However, natural hyaluronic acid has the following inherent defects: the intermolecular force between molecular chains is weak, resulting in insufficient mechanical properties of the formed three-dimensional network structure; it is easily degraded rapidly by hyaluronidase in vivo, and it is difficult to meet the mechanical support and long-term stability requirements for soft tissue repair.

[0003] In response to the above problems, chemical modification strategies have shown significant advantages: sodium periodate can introduce active aldehyde groups into the hyaluronic acid sugar ring through an oxidation reaction, and enhance tissue adhesion and self-healing ability through dynamic Schiff base reactions; methacrylic anhydride modification can provide photo-responsive double bond cross-linking sites, and a hyaluronic acid hydrogel with controllable cross-linking density can be constructed through ultraviolet light-initiated free radical polymerization. However, a single modification method also has obvious limitations: for example, the hydrogel formed by aldehyde-modified hyaluronic acid through dynamic Schiff base reactions has the defect of insufficient mechanical strength and is difficult to meet the mechanical requirements of load-bearing tissues; while the photo-cured methacryloylated hyaluronic acid-based hydrogel can form a high-strength covalent cross-linking network, but it loses its dynamic adaptability.

[0004] The dual-modification strategy provides an important technical path for constructing novel intelligent hydrogels. However, the existing preparation processes still have significant limitations: for example, traditional methods usually need to be carried out step by step, involving cumbersome processes such as two times of solution preparation, dialysis of the reaction solution, and freeze-drying, which seriously restricts the preparation efficiency of the dual-modification process; in addition, different functional groups may interfere with each other during the dual-modification process, inducing side reactions, and thus leading to differences in the structure and properties of the products. Therefore, developing a dual-modified hydrogel system with both an efficient preparation process and controllable properties has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of the present invention is to overcome the above technical deficiencies, and provide a one-step purification dual-modified hyaluronic acid hydrogel and a preparation method thereof, so as to solve the technical problems in the prior art that the preparation efficiency of hyaluronic acid hydrogels is relatively low or side reactions are easily induced, resulting in uncontrollable properties or poor stability of the hydrogels.

[0006] To achieve the above technical purpose, the technical solution provided by the present invention is: In a first aspect, the present invention provides a method for preparing a doubly modified hyaluronic acid hydrogel by one-step purification, comprising the following steps: Under light-shielded conditions, sodium periodate solution is added dropwise to a sodium hyaluronate solution for an aldehyde group formation reaction, and after quenching, an aldehyde group-modified hyaluronic acid reaction solution is obtained; The aldehyde group-modified hyaluronic acid reaction solution is cooled to 2-6 °C, and under light-shielded conditions, methylacrylic anhydride solution is added dropwise for an esterification reaction, controlling the reaction pH value to be 8-9. After the reaction ends, doubly modified hyaluronic acid is obtained through purification and drying; The doubly modified hyaluronic acid and a photoinitiator are dissolved in PBS buffer to form a precursor solution, and a doubly modified hyaluronic acid hydrogel is obtained through a photocrosslinking reaction.

[0007] In a second aspect, the present invention provides a doubly modified hyaluronic acid hydrogel prepared by the above preparation method.

[0008] Compared with the prior art, the beneficial effects of the present invention include: (1) The sodium hyaluronate selected in the present invention is a natural polysaccharide polymer, having excellent biocompatibility, biodegradability, and lubricating properties; (2) The present invention performs double modification of aldehyde group formation and methylacrylic anhydride modification on hyaluronic acid, which can optimize the two dialysis and freeze-drying steps required for the two modifications in the traditional method into a single step, significantly improving the double modification efficiency; Among them, the aldehyde group provides tissue adhesiveness, and the photosensitive group endows the material with gel-forming characteristics under light; (3) The present invention adopts the sequence of first aldehyde group functionalization and then methylacrylic anhydride modification, avoiding mutual interference between the two reactions, and by controlling conditions such as the reaction temperature (especially the esterification reaction temperature) and the pH value of the esterification reaction, the occurrence of side reactions is reduced; (4) The doubly modified hyaluronic acid hydrogel prepared by the present invention has a controllable gel time, excellent mechanical properties, anti-swelling property, and long-term stability, and at the same time exhibits good blood compatibility, having broad application prospects in the fields of tissue engineering, drug delivery, and soft tissue repair. Description of the Drawings

[0009] Figure 1 Nuclear magnetic spectrum analysis of the material prepared in Example 1; Figure 2 Shear viscosity test of the material prepared in Example 1; Figure 3 Gel time test of the materials prepared in Examples 1-4 and Comparative Examples 1-8; Among them, A corresponds to an AHM concentration of 2%, B corresponds to an AHM concentration of 3%, C corresponds to an AHM concentration of 4%, and D corresponds to an AHM concentration of 5%; Figure 4 Mechanical property test of the hydrogel obtained in Example 1; Figure 5Swelling property tests of the hydrogels obtained in Example 1, Comparative Example 1, and Comparative Example 2; Figure 6 Degradation property tests of the hydrogels obtained in Example 1, Comparative Example 1, and Comparative Example 2; Figure 7 Injectability tests of the hydrogel obtained in Example 1; A is the macroscopic view during injection, and B is the macroscopic view after injection; Figure 8 Blood compatibility tests of the hydrogel obtained in Example 1; A is the macroscopic view of the hemolysis experiment, and B is the hemolysis rate and coagulation index. Detailed implementation manners

[0010] In order to make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0011] In a first aspect, the present invention provides a preparation method for a one-step purified double-modified hyaluronic acid hydrogel, comprising the following steps: S1, under light-shielded conditions, drop a sodium periodate solution into a sodium hyaluronate solution to carry out an aldehyde group reaction, and obtain an aldehyde-functionalized hyaluronic acid reaction solution after quenching; S2, cool the aldehyde-functionalized hyaluronic acid reaction solution to 2 - 6 °C under an ice bath condition, and drop a methacrylic anhydride solution under light-shielded conditions to carry out an esterification reaction. During the reaction process, continuously control the pH value to be 8 - 9. After the reaction ends, obtain double-modified hyaluronic acid (AHM) through purification and drying; wherein the double modification refers to methacrylic anhydride modification and aldehyde group functionalization; S3, dissolve AHM and a photoinitiator in a PBS buffer solution to form a hydrogel precursor solution, and obtain a double-modified hyaluronic acid hydrogel through a photo-crosslinking reaction.

[0012] Preferably, in step S1, the sodium hyaluronate solution is prepared by dissolving sodium hyaluronate with a molecular weight of 200 - 400 kDa in deionized water, with a concentration of 4 - 6 w / v%, including but not limited to 4 w / v%, 5 w / v%, or 6 w / v%, etc.; the sodium periodate solution is prepared by dissolving sodium periodate in deionized water under light-shielded conditions; the molar ratio of the sugar unit of sodium hyaluronate to sodium periodate is (1 - 10):1, including but not limited to 1:1, 2:1, 4:1, 5:1, 6:1, 8:1, or 10:1, etc.; the volume ratio of the sodium hyaluronate solution to the sodium periodate solution is (2 - 6):1, including but not limited to 2:1, 3:1, 4:1, or 6:1, etc.

[0013] Preferably, in step S1, the temperature of the aldehyde group reaction is 20-30°C, the reaction time is 12-24 h, and the stirring speed is 400-800 r / min. Among them, the temperature of the aldehyde group reaction includes but is not limited to 20°C, 22°C, 25°C, 28°C, or 30°C, etc.; the reaction time includes but is not limited to 12 h, 14 h, 15 h, 18 h, 20 h, 22 h, or 24 h, etc. Quenching is to drop an excessive amount of ethylene glycol solution after the aldehyde group reaction is completed and continue stirring for 0.5-1 h to quench the unreacted sodium periodate, and the remaining reaction conditions are the same as those of the aldehyde group reaction.

[0014] Preferably, in step S2, the ratio of methacrylic anhydride to sodium hyaluronate is (0.5-2) mL:1 g, including but not limited to 0.5 mL:1 g, 1 mL:1 g, 1.5 mL:1 g, or 2 mL:1 g, etc.; the pH value is controlled to be 8.25-8.75 during the esterification reaction; the esterification reaction time is 2-6 h, including but not limited to 2 h, 4 h, 5 h, or 6 h, etc.; the stirring speed is 400-800 r / min.

[0015] Preferably, in step S2, purification is dialysis purification; drying is freeze-drying.

[0016] More preferably, in dialysis purification, the molecular weight cut-off of the dialysis bag is 14000 Da; the conditions for dialysis purification include: dialysis for 3-5 d, changing water 3-5 times a day; the conditions for freeze-drying include: the freezing time is 12-24 h, and the freeze-drying time is 36-72 h. Among them, freeze-drying can adopt conventional conditions to ensure drying, and specific limitations are not made here.

[0017] Preferably, in step S3, the photoinitiator is LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) or I2959 (2-hydroxy-2-methyl-1-[4-(2-hydroxyethoxy)phenyl]-1-propanone).

[0018] Preferably, in step S3, in the precursor solution, the concentration of AHM is 2-5 w / v%, including but not limited to 2 w / v%, 3 w / v%, 4 w / v%, or 5 w / v%, etc., and the concentration of the photoinitiator is 0.05-0.1 w / v%, including but not limited to 0.05 w / v%, 0.06 w / v%, 0.08 w / v%, or 0.1 w / v%, etc.

[0019] Preferably, in step S3, the conditions for the photocrosslinking reaction include: ultraviolet light irradiation for 5-60 s, including but not limited to 5 s, 10 s, 20 s, 25 s, 30 s, 40 s, 50 s, or 60 s, etc.; the wavelength of the ultraviolet light is 365 nm or 405 nm.

[0020] In a second aspect, the present invention provides a double-modified hyaluronic acid hydrogel prepared by the above preparation method.

[0021] The main mechanism of action and advantages of the present invention: (1) From the analysis of the reaction mechanism, if the process route of first performing methacrylic anhydride modification (esterification reaction) and then aldehyde group functionalization is adopted, the following technical defects may exist: The carbon-carbon double bond introduced by the esterification reaction may be oxidized and damaged by sodium periodate; The modified carbon-carbon double bond may produce steric hindrance effects, inhibiting the efficiency of subsequent aldehyde group reactions; The conditions required for the esterification reaction and the aldehyde group reaction are mutually contradictory, resulting in the generation of side reaction products; The present invention controls the reaction sequence, first performs the aldehyde group reaction and quenches the oxidant, and then performs the esterification reaction, avoiding the mutual interference of the two reactions. (2) The present invention controls the esterification reaction time within a relatively short range based on the following technical considerations: Relative to the feeding amount of sodium hyaluronate, methacrylic anhydride is in an excessive state, so there is no need for a long reaction time; Although extending the esterification reaction time can increase the grafting rate of methacrylic anhydride, too high a grafting rate will lead to a decrease in the stability of the obtained hydrogel. (3) The esterification reaction conditions also need to be controlled in the present invention: If the pH value of the reaction system is too high, the aldehyde group structure in the aldehyde group-modified hyaluronic acid will be damaged; If the pH value of the reaction system is too low, side products of the esterification reaction will be generated; Similarly, the ice bath condition is also to reduce the occurrence of side reactions.

[0022] In summary, the present invention adopts a step-by-step reaction strategy of first aldehyde group modification and then methacrylic anhydride modification, effectively avoiding the mutual interference of the two functional group modification reactions by optimizing the reaction sequence. Specifically: After the aldehyde group reaction is completed, ethylene glycol is used for selective quenching, and this quenching process will not affect the subsequent methacrylic anhydride modification reaction; The esterification modification process of methacrylic anhydride will not have an adverse effect on the formed aldehyde group functional groups either. In the present invention, by controlling the reaction conditions, including: reaction sequence, reaction temperature and pH value, etc., it plays a role in avoiding the mutual interference of the two reactions (sequence) and reducing the occurrence of side reactions (ice bath and pH conditions).

[0023] The present invention will be further described in detail below through specific examples; Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. In the following implementation process, unless otherwise specified, the raw materials are purchased from commercial channels.

[0024] Example 1 A preparation method for a one-step purified double-modified hyaluronic acid hydrogel, comprising the following steps: S1, 6 g (15 mmol) of sodium hyaluronate was dissolved in 120 mL of deionized water to obtain a sodium hyaluronate solution; under light-shielded conditions, 0.8205 g (3.75 mmol) of sodium periodate was dissolved in 30 mL of deionized water to obtain a sodium periodate solution. At this time, the molar ratio of the sugar units of sodium hyaluronate to sodium periodate was approximately 4:1; under light-shielded conditions, the sodium periodate solution was added dropwise to the sodium hyaluronate solution, and the reaction was continuously carried out at room temperature (25 °C) for 24 h. After the reaction was completed, an excess of ethylene glycol was added and the reaction was continued for 1 h to quench the unreacted sodium periodate, obtaining an aldehyde-modified hyaluronic acid reaction solution; S2, The aldehyde-modified hyaluronic acid reaction solution was cooled to 4 °C, and 6 mL of methacrylic anhydride was added dropwise under light-shielded conditions. The pH was continuously adjusted in the range of 8.25 - 8.75, and the esterification reaction was carried out for 2 h to obtain a doubly modified hyaluronic acid reaction solution. It was filled into a dialysis bag with a molecular weight cut-off of 14,000 Da and dialyzed in deionized water, changing the water 4 - 5 times a day. After dialysis for 3 days, it was freeze-dried to obtain purified AHM; S3, The purified AHM and the photoinitiator LAP were respectively dissolved in PBS buffer at concentrations of 5 w / v% and 0.05 w / v%. After mixing evenly, they were injected into a polytetrafluoroethylene mold with a diameter of 10 mm and a height of 5 mm. After being irradiated with 405 nm ultraviolet light for 40 s, a gel was formed to obtain a doubly modified hyaluronic acid hydrogel.

[0025] Example 2 Compared with Example 1, the only difference is that: in step S3, the concentration of the doubly modified hyaluronic acid is 4 w / v%, and the other steps and conditions are the same as those in Example 1.

[0026] Example 3 Compared with Example 1, the only difference is that: in step S3, the concentration of the doubly modified hyaluronic acid is 3 w / v%, and the other steps and conditions are the same as those in Example 1.

[0027] Example 4 Compared with Example 1, the only difference is that: in step S3, the concentration of the doubly modified hyaluronic acid is 2 w / v%, and the other steps and conditions are the same as those in Example 1.

[0028] Comparative Example 1 Compared with Example 1, the only difference is that: the time of the esterification reaction was adjusted to 4 h, and the other steps and conditions are the same as those in Example 1.

[0029] Comparative Example 2 Compared with Example 1, the only difference is that: the time of the esterification reaction was adjusted to 6 h, and the other steps and conditions are the same as those in Example 1.

[0030] Comparative Example 3 Compared with Example 2, the only difference is that the time of the esterification reaction is adjusted to 4 h, and the other steps and conditions are the same as those in Example 2.

[0031] Comparative Example 4 Compared with Example 2, the only difference is that the time of the esterification reaction is adjusted to 6 h, and the other steps and conditions are the same as those in Example 2.

[0032] Comparative Example 5 Compared with Example 3, the only difference is that the time of the esterification reaction is adjusted to 4 h, and the other steps and conditions are the same as those in Example 3.

[0033] Comparative Example 6 Compared with Example 3, the only difference is that the time of the esterification reaction is adjusted to 6 h, and the other steps and conditions are the same as those in Example 3.

[0034] Comparative Example 7 Compared with Example 4, the only difference is that the time of the esterification reaction is adjusted to 4 h, and the other steps and conditions are the same as those in Example 4.

[0035] Comparative Example 8 Compared with Example 4, the only difference is that the time of the esterification reaction is adjusted to 6 h, and the other steps and conditions are the same as those in Example 4.

[0036] Comparative Example 9 Compared with Example 1, the only difference is that the pH value in Step S2 is adjusted to 7 - 7.5, and the other steps and conditions are the same as those in Example 1.

[0037] Comparative Example 10 Compared with Example 1, the only difference is that the pH value in Step S2 is adjusted to 10 - 10.5, and the other steps and conditions are the same as those in Example 1.

[0038] It was found that too high or too low pH value was not conducive to obtaining the target product. Under too alkaline conditions, the aldehyde group would be destroyed; when the pH was too low, by-products of the esterification reaction would be generated.

[0039] Performance Test and Result Analysis 1. NMR Spectra Analysis Test Method: Sodium hyaluronate and the purified AHM obtained in Step S2 were subjected to 1 1H NMR detection by a nuclear magnetic resonance spectrometer (Advance III, Bruker, Switzerland). First, 5 - 10 mg of the dried sample was weighed and fully dissolved in 0.5 mL of deuterated water (D2O), and it was ensured that the solution was clear and free of impurities. Then, the solution was transferred to a nuclear magnetic resonance tube and tested and analyzed by the nuclear magnetic resonance spectrometer at 25 °C.

[0040] Test results: In Figure 1 , the broad signal peak between 3.0 ppm and 4.7 ppm is considered as the different proton peaks in the hyaluronic acid sugar units. Several signal peaks between 4.9 ppm and 5.1 ppm are attributed to the newly generated aldehyde groups, confirming the formation of aldehyde groups. In addition, two new peaks at 5.7 ppm and 6.1 ppm respectively correspond to the two methylene proton peaks on RC=CH2, originating from methacrylic anhydride, confirming the successful grafting of double bonds.

[0041] 2. Shear viscosity measurement Test method: Use a rotational rheometer (HAAKE RheoStress 6000, Thermo Fisher, USA) to conduct shear tests on sodium hyaluronate solution and double-modified hyaluronic acid solution. First, weigh an appropriate amount of sodium hyaluronate sample and the purified AHM sample obtained in step S2, dissolve them in ultrapure water, and prepare a solution with a final concentration of 5 w / v%. Subsequently, place the solution in a refrigerator at 4 °C overnight to remove air bubbles. Conduct shear viscosity tests on it at room temperature of 25 °C, and set the gap between the upper and lower plates to 1 mm during the test. Record the data after the values are stable.

[0042] Test results: It can be seen from Figure 2 that as the shear rate increases, the viscosity of the solution gradually decreases and tends to be stable, which conforms to the shear thinning phenomenon of non-Newtonian fluids. The shear viscosity of the AHM solution (6.835 mPa·s) is significantly lower than that of the sodium hyaluronate solution (3589 mPa·s), indicating that the double modification significantly reduces the molecular weight of sodium hyaluronate.

[0043] 3. Gel time test Test method: Determine the gel time through the vial inversion experiment. The specific operation is as follows: Take 1 mL of the precursor solutions in Examples 1-4 and Comparative Examples 1-8 respectively and place them into 5 mL round-bottom centrifuge tubes, and continuously irradiate them with 405 nm ultraviolet light until the gel does not flow after inverting the centrifuge tubes, and record the gel time at this time (the recorded time is taken as an integer value). Set 3 parallel samples for each group of samples, and take the average value of the final results.

[0044] Test results: Figure 3 And Table 1 shows the gel times of the AHM solutions obtained in Examples 1-4 and Comparative Examples 1-8 with different concentrations.

[0045] Table 1 Gel times of Examples 1-4 and Comparative Examples 1-8

[0046] From Table 1 and Figure 3It can be observed that as the solution concentration increases, the gelation times of the hydrogel precursor solutions in each example and the comparative examples all shorten and gradually tend to be stable. The gelation time significantly decreases from 22 s at a concentration of 2 w / v% in Example 4 to 9 s at a concentration of 5 w / v% in Example 1. This is because there is a certain correlation between the gelation time of the hydrogel and the solution concentration. A higher solution concentration provides more gelation sites and chain entanglements, so the gelation time is shorter.

[0047] Theoretically, extending the reaction time can increase the grafting rate of methacrylic anhydride, thereby providing more crosslinking sites and shortening the gelation time. However, in the three groups of hydrogels of Example 1, Comparative Example 1, and Comparative Example 2 (the same applies to other groups), as the methacrylation time increases, the gelation time actually prolongs. The gelation times of Examples 1-4 are all lower than those of the corresponding comparative examples in the same group. This may be due to the poor water solubility of the final grafted product. An excessively high grafting rate will cause the product to precipitate, thereby reducing the actual concentration. In addition, the precipitated product may float in the precursor solution, blocking light, thus affecting the photo-crosslinking process. The changes in the gelation times of these three groups may be the combined result of the decrease in the actual solution concentration and the hindrance of the photo-crosslinking process.

[0048] 4. Mechanical property testing of hydrogels Testing method: The mechanical properties of the double-modified hyaluronic acid hydrogel were determined by a Mest universal testing machine (CMT-10, Instron MTS, USA). Cylindrical hydrogel samples were prepared in a mold (diameter 10 mm, height 5 mm). Using a load of 50 N, these samples were tested for compressive capacity at a constant loading speed of 1 mm / min until the samples ruptured. The stress-strain curves were recorded and plotted using Origin software. The compression modulus (E) of the hydrogel was calculated by calculating the slope of the first 30% of the curve. Three parallel samples were set for each group of samples.

[0049] Testing results: Figure 4 The representative stress-strain curve and modulus of the AHM hydrogel in Example 1 are shown. The fracture strain and fracture strength of the hydrogel are 34% and 70 kPa respectively, and the modulus is 143 kPa. This indicates that the hydrogel has good supporting ability.

[0050] 5. Swelling property testing of hydrogels Testing method: The swelling properties of the AHM hydrogel were determined by the gravimetric method. Hydrogels were prepared in a mold. After demolding, the excess water on the surface was carefully wiped off with weighing paper, and then weighed and the initial mass W0 was recorded. Subsequently, each hydrogel was placed in a 24-well plate containing 2 mL of PBS buffer, and the whole was placed in a thermostatic shaker (37 °C, 100 rpm). The hydrogels were taken out at different time points and the residual water on the surface was wiped off with filter paper, and the weight W was recorded.t , the swelling ratio is calculated according to Equation (1). Three parallel samples are set for each group of samples, and the final result is the average value of them.

[0051] (1) Test results: As Figure 5 shown, the swelling ratios of the hydrogels obtained in Example 1 all increased slowly over time and basically reached swelling equilibrium after being soaked in PBS for 24 h, and the final equilibrium swelling ratio was 7.2%. For Comparative Example 1 and Comparative Example 2, the increase in the esterification time had little effect on the equilibrium swelling ratio, which were 8.2% and 9% respectively. The hydrogels of all components had relatively low swelling ratios (less than 15%), indicating that these hydrogels had good anti-swelling ability and were suitable for applications in fields such as tissue engineering.

[0052] 6. Degradation performance test of hydrogels Test method: The degradation performance of the AHM hydrogel was determined by the gravimetric method. The hydrogel was prepared in a mold. After demolding, the excess moisture on the surface was carefully wiped off with weighing paper, and its initial mass W0 was recorded after freeze-drying. Similar to the swelling performance test, the freeze-dried hydrogel was placed in a 24-well plate containing 2 mL of PBS buffer solution, and the whole was placed in a thermostatic shaker (37 °C, 100 rpm). Samples were taken out at the established time points (7, 14, 21, 28 d), freeze-dried again and their final weights (W t ) were recorded. The degradation rate was calculated according to Equation (2). Three parallel samples were set for each group of samples, and the final result was the average value of them.

[0053] (2) Test results: As Figure 6 shown, the degradation curves of the hydrogels prepared in Example 1, Comparative Example 1 and Comparative Example 2 increased with time, and the degradation rates on the 28th day were 48.85%, 56.27% and 81.04% respectively. This indicates that with the increase in the esterification time, the stability of the hydrogel decreased, which had a significant impact on the degradation rate.

[0054] In summary, compared with Example 1, the gel time of Comparative Example 1 was prolonged, and at the same time, the swelling ratio and degradation rate increased; the gel time of Comparative Example 2 was longer, and at the same time, the swelling ratio and degradation rate were higher. Therefore, in the present invention, the esterification reaction time is preferably 2 - 6 h, more preferably 2 - 4 h; the concentration of AHM is preferably 2 - 5 w / v%, more preferably 3 - 5 w / v%.

[0055] 7. Injectability test of hydrogels Testing method: To evaluate the injectability of the AHM hydrogel, an auxiliary test was carried out using a disposable medical syringe (1 mL, 26G×1 / 2''). The AHM precursor solution of Example 1 was transferred into a 1 mL syringe and crosslinked into a gel inside the syringe by ultraviolet light irradiation. If the hydrogel sample could be continuously and evenly pushed out from the syringe, it was considered to have good injectability. To enhance the color contrast, a small amount of methyl orange was added to the precursor solution to achieve a color development effect.

[0056] Testing results: The macroscopic injectability experimental results of the AHM hydrogel of Example 1 are as Figure 7 shown. Figure 7 Figure A therein shows the adhesion of the hydrogel during the injection process, indicating that the hydrogel could be continuously pushed out through a 26G needle syringe after preliminary crosslinking. Figure 7 The macroscopic view in Figure B therein indicates that the hydrogel could push out complex words such as "MH". Generally speaking, the AHM hydrogel showed good injectability and could be applied by injection.

[0057] 8. Blood compatibility test of the hydrogel Testing method: Red blood cells were extracted from the blood of sterile Kunming mice by centrifugation, and the red blood cells were diluted to 2% with 0.9% physiological saline to prepare a red blood cell suspension. 100 mg of the hydrogel was pre-placed in 20 mL of 0.9% physiological saline and incubated in a thermostatic shaker (37 °C, 100 rpm) for 24 h to obtain an extract of the hydrogel. 100 μL of each extract was taken and mixed with 900 μL of the red blood cell suspension in a 1.5 mL centrifuge tube, and then incubated in the thermostatic shaker for another 1 h. 0.9% physiological saline was used to replace the extract as the negative control group, and ultrapure water was used as the positive control group. After the incubation, all centrifuge tubes were centrifuged at 1500 rpm for 5 to 10 minutes, 100 μL of the supernatant was transferred into a 96-well plate, and the absorbance value at a wavelength of 540 nm was measured using an enzyme-linked immunosorbent assay (Multiskan FC, Thermo Scientific, USA), and the hemolysis ratio (HR) was calculated by Equation (3). Among them, A Sample 、A Positive 、A Negative represent the absorbance values of the test group (AHM), the positive control group (+) and the negative control group (-) respectively. Three parallel samples were set for each group of samples.

[0058] (3) Transfer 1 mL of the hydrogel precursor solution into a 5 mL round-bottom centrifuge tube, crosslink it into a gel inside, and then incubate it in a constant temperature shaker (37 °C, 100 rpm) for stabilization. Drop 20 μL of anticoagulated mouse whole blood evenly on the upper surface of the hydrogel and continue to incubate it in the constant temperature shaker for 15 min. Subsequently, add 2 μL of 0.1 M CaCl2 solution to the blood to initiate the coagulation mechanism, and place the centrifuge tube in the constant temperature shaker and continue to incubate for 5 min. Slowly add 2 mL of ultrapure water to remove the uncoagulated blood cells without affecting the blood clot. Next, transfer 100 μL of the supernatant into a 96-well plate, measure its absorbance value at a wavelength of 540 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the BCI index through Equation (4). Among them, A Sample and A Control represent the absorbance values of the test group and the blank control group, respectively. Three parallel samples are set for each group of samples.

[0059] (4) Test results: As can be seen from A in Figure 8 , except for the positive control group (+), the AHM hydrogel extract group and the negative control group (-) showed light yellow color and were accompanied by red blood cell precipitation, and no obvious hemolysis occurred. At the same time, the hemolysis rate of the hydrogel was 1.15%, which was lower than 5%. This indicates that the hydrogel material caused less damage to red blood cells and had good blood compatibility. In addition, the coagulation index mainly reflects the coagulation effect of the material. The lower the coagulation index value, the better the coagulation effect of the material; Figure 8 B in

[0060] also showed that the coagulation index value of the hydrogel was 29.07%. The results show that the AHM hydrogel material of the present invention has a good coagulation effect and is expected to quickly adhere to and seal the wound after in-situ filling of tissue engineering defects.

[0061] The specific embodiments of the present invention described above do not constitute a limitation on the protection scope of the present invention. Any other corresponding changes and deformations made according to the technical concept of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. A preparation method of a one-step purified double-modified hyaluronic acid hydrogel, characterized in that, It includes the following steps: Under light-shielding conditions, sodium periodate solution is added dropwise to the sodium hyaluronate solution for aldehyde group reaction, and after quenching, an aldehyde group-modified hyaluronic acid reaction solution is obtained; The aldehyde group-modified hyaluronic acid reaction solution is cooled to 2-6°C, and methacrylic anhydride solution is added dropwise under light-shielding conditions for esterification reaction. The reaction pH value is controlled to be 8-9. After the reaction ends, double-modified hyaluronic acid is obtained through purification and drying; The double-modified hyaluronic acid and a photoinitiator are dissolved in PBS buffer solution to form a precursor solution, and a double-modified hyaluronic acid hydrogel is obtained through photo-crosslinking reaction.

2. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The sodium hyaluronate solution is prepared by dissolving sodium hyaluronate with a molecular weight of 200-400 kDa in deionized water; the sodium periodate solution is prepared by dissolving sodium periodate in deionized water under light-shielding conditions.

3. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The concentration of the sodium hyaluronate solution is 4-6 w / v%, the molar ratio of the sugar unit of sodium hyaluronate to sodium periodate is (1-10):1, and the volume ratio of the sodium hyaluronate solution to the sodium periodate solution is (2-6):

1.

4. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The temperature of the aldehyde group reaction is 20-30°C, the reaction time is 12-24 h, and the stirring speed is 400-800 r / min.

5. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The quenching is to add an excessive amount of ethylene glycol solution after the aldehyde group reaction ends, and continue stirring for 0.5-1 h.

6. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, wherein, The ratio of methacrylic anhydride to sodium hyaluronate is (0.5-2) mL:1 g; during the esterification reaction, the pH value is controlled to be 8.25-8.75, the esterification reaction time is 2-6 h, and the stirring speed is 400-800 r / min.

7. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The purification is dialysis purification; the drying is freeze-drying.

8. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, characterized in that, The photoinitiator is LAP or I2959; In the precursor solution, the concentration of the double-modified hyaluronic acid is 2-5 w / v%, and the concentration of the photoinitiator is 0.05-0.1 w / v%.

9. The preparation method of the one-step purified double-modified hyaluronic acid hydrogel according to claim 1, wherein The conditions of the photo-crosslinking reaction include: ultraviolet light irradiation for 5-60 s.

10. A double-modified hyaluronic acid hydrogel prepared by the preparation method according to any one of claims 1-9.

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