Method for preparing a responsive dynamic hydrogel and use thereof

Abstract

The application discloses a preparation method of a responsive dynamic hydrogel, and the method comprises the following steps: firstly, dispersing sodium alginate and then performing a directional oxidation reaction with a sodium periodate aqueous solution to obtain oxidized sodium alginate containing an aldehyde group, and then dissolving the oxidized sodium alginate in a hot polyvinyl alcohol solution to obtain a precursor mother liquor A; secondly, dissolving a water-soluble chitosan derivative in deionized water, adding aminobenzene boronic acid small molecules, and then heating to obtain a precursor mother liquor B; and thirdly, mixing the precursor mother liquor A and the precursor mother liquor B to obtain the dynamic hydrogel. The precursor mother liquor A is coated on a medical material substrate, and then the precursor mother liquor B is coated on the medical material substrate. The aldehyde group and the amine group are utilized to form an imine bond crosslinking, and meanwhile, the benzene boronic acid group and the vicinal diol are utilized to form a borate ester bond crosslinking, so that the prepared hydrogel has a rapid gelation capacity, pH and ROS responsiveness and a suitable elastic modulus. After the hydrogel is applied to the medical material substrate, the bioreactivity and the interfacial mechanical softness of the medical material substrate are improved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medical coatings, and particularly relates to a preparation method of a responsive dynamic hydrogel and application thereof. BACKGROUND

[0002] When biomedical materials are applied to implantation or interventional therapy scenarios, they will often be in direct contact with human soft tissues. At present, common biomedical materials covering metals, ceramics, polymers and other types have generally poor compatibility with human soft tissues in terms of mechanical properties and other physical and chemical properties. This mismatch can easily induce inflammation and scar tissue formation and other immune responses in the local body. For example, the hydrophobic surface of a vascular stent often causes proteins and platelets to accumulate and adhere to the stent surface, which can cause restenosis of the blood vessel and even thrombosis, seriously affecting the treatment effect and patient health and safety.

[0003] Hydrogels, as a class of high-water-content solid-like materials, have unique soft mechanical properties and excellent biocompatibility, and have great application potential in the biomedical field. They are a functional coating material that has attracted much attention. When hydrogel coatings are constructed on biomedical material substrates, the three-dimensional network architecture formed by cross-linking has obvious advantages, providing convenience for the loading of various drugs and facilitating efficient drug loading, and can effectively optimize the biological functionality of implantation and interventional materials. Among them, hydrogels constructed by polymer networks based on dynamic covalent bonds have unique performance. On the one hand, they exhibit shear-thinning characteristics under stress response, facilitating deformation adaptation in different medical applications, and at the same time exhibiting the ability to respond to specific chemical stimuli. Hydrogels cross-linked by borate ester bonds can produce network de-cross-linking behavior in response to glucose and reactive oxygen species (ROS), which meets the local drug controlled-release treatment needs of diabetic and cancer patients. Hydrogels cross-linked by imine bonds are sensitive to pH changes and can release loaded drugs in a local physiological acidic environment. In addition, since dynamic covalent bonds have the advantages of robustness and reversibility, hydrogels cross-linked by them can be used as coatings for biomedical materials or medical devices. On the one hand, they can give the coating appropriate mechanical strength and adaptability, ensuring that the coating can function stably under complex stress conditions. On the other hand, their self-healing properties can effectively repair micro-damage that may occur in the coating, greatly expanding the range of practical application scenarios and prolonging the service life of the coating. Finally, the rapid cross-linking kinetics of dynamic covalent bonds is conducive to the rapid formation of hydrogel coatings, fully meeting the needs of rapid preparation, long-term, stable and efficient use in biomedical scenarios.

[0004] Therefore, constructing a responsive hydrogel coating based on dynamic borate ester bond and imine bond crosslinking on the surface of medical materials to optimize the mechanical adaptation degree of the materials, strengthen the biocompatibility, and improve the hydrophilic and hydrophobic interface properties has become a very key front development trend in the field of medical coating technology, and has far-reaching significance for improving the clinical application performance of biomedical materials and ensuring the prognosis of patients. SUMMARY

[0005] The technical problem to be solved by the present application is to provide a preparation method of a responsive dynamic hydrogel coating to solve the problems of great difficulty, high cost and lack of functionality of the hydrogel coating prepared by the existing medical materials and devices.

[0006] To solve the above technical problems, the technical scheme adopted by the present application is as follows: a preparation method of a responsive dynamic hydrogel, characterized in that the method comprises the following steps:

[0007] Step one, preparation of hydrogel precursor mother liquor A: sodium alginate is dispersed in anhydrous ethanol, and a directional oxidation reaction is carried out under light shielding conditions using a sodium periodate aqueous solution. After the reaction is terminated, purified aldehyde group-containing oxidized sodium alginate is obtained. Then, the aldehyde group-containing oxidized sodium alginate is dissolved in a hot polyvinyl alcohol solution to obtain precursor mother liquor A.

[0008] Step two, preparation of hydrogel precursor mother liquor B: water-soluble chitosan derivatives are dissolved in deionized water, then amino phenyl boronic acid micromolecules are added, and heated and stirred to obtain precursor mother liquor B.

[0009] Step three, preparation of hydrogel: the precursor mother liquor A obtained in step one and the precursor mother liquor B obtained in step two are rapidly stirred and mixed uniformly at a volume ratio of 1:1-2 to obtain a transient gelation, pH and ROS responsive dynamic hydrogel.

[0010] Generally, in step one of the present application, sodium alginate is added to rapidly stirred anhydrous ethanol, and then a sodium periodate aqueous solution is added dropwise under light shielding conditions. After the dropwise addition is completed, the directional oxidation reaction is carried out at room temperature for 6 hours. After the reaction is completed, ethylene glycol is added to terminate the reaction, and the stirring at room temperature is continued overnight. Finally, the obtained suspension is transferred to a large amount of stirred anhydrous ethanol, the precipitate is collected by filtration, then the precipitate is dissolved in deionized water and dialyzed in deionized water, and finally freeze-dried to obtain aldehyde group-containing oxidized sodium alginate.

[0011] The content of aldehyde groups in the oxidized sodium alginate is determined by the hydroxylamine hydrochloride titration method described in the patent with application number 202311594957.9. Generally, the content of aldehyde groups in the oxidized sodium alginate is 6.5 mmol / g to 8.1 mmol / g.

[0012] The relative molecular weight of the oxidized sodium alginate is determined by gel permeation chromatography (GPC). Generally, the average molecular weight of the oxidized sodium alginate is 120 kDa to 154 kDa, and the molecular weight distribution (DPI) is 1.29 to 1.38.

[0013] The preparation method of the responsive dynamic hydrogel described above, characterized in that the concentration of the sodium alginate dispersed in anhydrous ethanol in step one is 0.1 g / mL to 0.2 g / mL. Generally, heating magnetic stirring is used for dispersion, and the dispersion temperature is 40°C, and the dispersion time is 30 min to 60 min, to obtain a uniform dispersion of the sodium alginate in anhydrous ethanol suspension without massive aggregation.

[0014] The preparation method of the responsive dynamic hydrogel described above, characterized in that the molar ratio of sodium periodate to sodium alginate monomer units in the sodium periodate aqueous solution in step one is 0.5:1 to 1.2:1.

[0015] The preparation method of the responsive dynamic hydrogel described above, characterized in that the method for terminating the reaction in step one is to add ethylene glycol, and the molar ratio of ethylene glycol to sodium periodate in the sodium periodate aqueous solution is 1.5:1 to 1.2:1.

[0016] The preparation method of the responsive dynamic hydrogel described above, characterized in that the method for purifying in step one is to transfer the reaction system solution drop by drop to 2 to 3 times the amount of stirring anhydrous ethanol, then collect the precipitate by filtration, dissolve the precipitate in deionized water and dialyze in deionized water, and then freeze-dry. Generally, the precipitated white precipitate is collected by filtration, the solid precipitate is collected after multiple suction filtration, then the solid precipitate is dissolved in deionized water, and the dialysis bag is filled with deionized water and dialyzed for 1 to 2 weeks, and then freeze-dried to obtain white cotton-like oxidized sodium alginate.

[0017] The preparation method of the responsive dynamic hydrogel described above, characterized in that the concentration of the total polymer in the precursor mother liquor A in step one includes oxidized sodium alginate containing aldehyde groups and polyvinyl alcohol, the concentration of polyvinyl alcohol is 0.1 g / mL, and the mass ratio of oxidized sodium alginate containing aldehyde groups to polyvinyl alcohol is 5:2.

[0018] Generally, the polyvinyl alcohol is fully swelled in cold water, heated and stirred in a 95℃ metal bath for 2h to obtain a transparent, uniform polyvinyl alcohol solution with a mass volume concentration of 10%; then, under stirring in an 80℃ metal bath, the aldehyde group-containing oxidized sodium alginate is added and stirred for 30min to obtain a yellowish, translucent, uniform solution, i.e., precursor mother liquor A.

[0019] The method for preparing the responsive dynamic hydrogel has the characteristics that in step two, the water-soluble chitosan derivative is carboxymethyl chitosan, carboxylated chitosan, chitosan quaternary ammonium salt, chitosan hydrochloride or chitosan lactate, and the concentration in deionized water is 0.02g / mL; the amino phenylboronic acid small molecule is 2-amino phenylboronic acid, 3-amino phenylboronic acid or 4-amino phenylboronic acid, and the concentration after addition is 0.018g / mL.

[0020] Generally, the water-soluble chitosan derivative is fully swelled in cold water, heated and stirred in a 50℃ metal bath for 2h to obtain a transparent, uniform solution; then, under stirring in a 50℃ metal bath, the amino phenylboronic acid small molecule is added and stirred for 2h, and the reaction is continued under stirring in a 60℃ metal bath for 6h to obtain a transparent, uniform solution, i.e., precursor mother liquor B.

[0021] Meanwhile, the application also discloses an application of the responsive dynamic hydrogel prepared by the method, which has the characteristics that the precursor mother liquor A obtained in step one is coated on a medical material substrate, and then the precursor mother liquor B obtained in step two is coated on the precursor mother liquor A to obtain a dynamic hydrogel coating layer with instantaneous gelation, pH and ROS responsiveness on the surface of the medical material.

[0022] The application has the characteristics that the coating methods of the precursor mother liquor A and the precursor mother liquor B are derived from brushing, scraping, solution casting, dipping, spinning, spraying, inkjet / 3D printing.

[0023] The application has the characteristics that the medical material substrate is derived from metallic titanium, magnesium, zinc, iron and alloys thereof, ceramic hydroxyapatite, polypropylene, polyvinyl chloride high polymer medical materials and devices.

[0024] Compared with the prior art, the application has the following advantages:

[0025] 1. The application adopts the o-diol of the benzene boronic acid of the small molecule of amino benzene boronic acid and the water-soluble chitosan derivative to graft on the chitosan derivative chain by forming borate ester bond, then mixed with the hot solution containing aldehyde group of oxidized sodium alginate and polyvinyl alcohol, so that the aldehyde group of oxidized sodium alginate is crosslinked with the amine group of chitosan and grafted / ungrafted amino benzene boronic acid to form imine bond network, and the ungrafted amino benzene boronic acid and the benzene boronic acid of borate ester bond dynamic dissociation further rapidly form borate ester bond crosslinking with the o-diol of polyvinyl alcohol and oxidized sodium alginate, under the effect of the above multiple crosslinking effect, promote rapid crosslinking to form instantaneous hydrogel, so that the dynamic hydrogel with instantaneous gelation, pH and ROS responsiveness is obtained.

[0026] 2. The dynamic hydrogel prepared by the application is crosslinked based on dynamic borate ester bond and imine bond, can respond to ROS (reactive oxygen) and pH, can be used for inflammatory and low-pH treatment sites, and can be used for controlled release of drug loading, thereby improving the treatment effect of medical materials.

[0027] 3. The functional polymer for preparing the dynamic hydrogel of the application only needs to synthesize oxidized sodium alginate, and the synthesis process is easy to realize and low in cost, thereby reducing the preparation cost of the dynamic hydrogel.

[0028] 4. The main polymers for preparing the dynamic hydrogel of the application are biologically derived sodium alginate and chitosan derivatives, and polyvinyl alcohol with good biocompatibility, and all of them can be degraded to a certain extent under physiological conditions, which is beneficial to the dynamic hydrogel as an inert or degradable biomedical material coating, and improves the biocompatibility and safety of the material matrix.

[0029] 5. The dynamic hydrogel coating of the application is formed by contact of two precursor mother solutions with different viscosities, the precursor mother solution A containing polyvinyl alcohol and oxidized sodium alginate with high viscosity and good adhesion is first attached to the surface of the medical material matrix, then the precursor mother solution B containing chitosan and amino benzene boronic acid with low viscosity is spread on it and quickly gels with the precursor mother solution A to form a non-flowing hydrogel coating, the hydrogel is formed by instantaneous rapid crosslinking, the preparation efficiency of the hydrogel coating on the surface of the medical material is improved, and various coating processes such as brushing, scraping, solution casting, dipping, spinning, spraying, inkjet / 3D printing can be applied, and the operation range is wide.

[0030] 6. The dynamic hydrogel coating of the application can adjust the dynamic mechanical properties and elastic modulus of the coating by adjusting the composition or concentration of the preparation mother solution, and shows adjustable shear thinning behavior, and can be constructed by injection, printing and other methods after gel formation, and can realize the construction of complex and irregular hydrogel coatings of various medical materials or devices.

[0031] 7、The prepared dynamic hydrogel is based on reversible dynamic borate ester bonds and imine bond crosslinking, exhibits certain self-healing performance, is beneficial to self-repairing as a coating when suffering external stress damage, and has an important role in prolonging the service life of the hydrogel coating.

[0032] 8、The dynamic hydrogel coating of the application can be prepared on various medical material or device substrates, has a wide application range, and has good medical industry application prospect due to the strong operability and good repeatability of the coating preparation method.

[0033] The technical solutions of the application are described in further detail below by means of the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 It is a Fourier infrared spectrum of sodium alginate and oxidized sodium alginate in the inventive example 1.

[0035] Figure 2 It is a pH and ROS response gel-dissolution transition diagram of the dynamic hydrogel prepared in the inventive example 2.

[0036] Figure 3 It is a rheological test frequency scanning result diagram of the dynamic hydrogel prepared in the inventive example 2 and the inventive example 4.

[0037] Figure 4 It is a rheological test strain scanning result diagram of the dynamic hydrogel prepared in the inventive example 2 and the inventive example 4.

[0038] Figure 5 It is the NIN / 3T3 cell survival rate of the dynamic hydrogel prepared in the inventive examples 1-3 after 48h treatment.

[0039] Figure 6 It is a SEM diagram of the dynamic hydrogel prepared in the inventive examples 1-3.

[0040] Figure 7 It is a process schematic diagram of the application of the responsive dynamic hydrogel to a medical material surface coating.

[0041] Figure 8 It is a SEM diagram of the magnesium alloy WE43 round sheet surface after alkali heat treatment in the inventive example 15.

[0042] Figure 9 It is a SEM diagram of the magnesium alloy WE43 round sheet surface after forming a dynamic hydrogel coating in the inventive example 15.

[0043] Figure 10 It is a contact angle test result of the magnesium alloy WE43 round sheet surface after alkali heat treatment and forming a dynamic hydrogel coating in the inventive example 13, the inventive example 14 and the inventive example 15. DETAILED DESCRIPTION

[0044] The preparation method of the responsive dynamic hydrogel of the present application is described in detail by Examples 1-12.

[0045] Example 1

[0046] This example includes the following steps:

[0047] Step one, preparation of hydrogel precursor mother liquor A: 20 g of sodium alginate was dispersed in 100 mL of anhydrous ethanol and stirred in a 40°C water bath for 60 min to obtain a uniformly dispersed turbid solution. 100 mL of 0.108 g / mL sodium periodate aqueous solution was added dropwise to the turbid solution under dark conditions, the molar ratio of sodium periodate to sodium alginate monomer units was 0.5:1. After the addition was completed, the directional oxidation reaction was continued at room temperature for 6 h under dark conditions. After the reaction was completed, 4.7 g of ethylene glycol was added to terminate the reaction, the molar ratio of ethylene glycol to sodium periodate in the sodium periodate aqueous solution was 1.5:1, and the reaction system was stirred overnight at room temperature. The solution was transferred dropwise to 600 mL of stirring anhydrous ethanol, then the precipitate was collected by filtration, dissolved in deionized water and loaded into a dialysis bag (MWCO: 8000 Da-14000 Da), dialyzed in deionized water for 2 weeks, and then freeze-dried after completion. Oxidized sodium alginate containing aldehyde groups was obtained. The aldehyde group concentration of the oxidized sodium alginate was 6.5 mmol / g, the number average molecular weight was 154 kDa, and the molecular weight distribution DPI was 1.29. 0.2 g of oxidized sodium alginate containing aldehyde groups was dissolved in 5 mL of 10% polyvinyl alcohol aqueous solution at 80°C in a stirring state, and stirred for 30 min to obtain precursor mother liquor A.

[0048] Step two, preparation of hydrogel precursor mother liquor B: 0.1 g of carboxymethyl chitosan was dissolved in 5 mL of deionized water, swelled for 0.5 h, then dissolved by stirring at 50°C for 2 h, then 0.09 g of 3-aminobenzoic acid was added, and the mixture was stirred at 50°C for 2 h, then at 60°C for 6 h, and the stirring process was sealed or protected by nitrogen. Precursor mother liquor B was obtained.

[0049] Step three, preparation of hydrogel: precursor mother liquor A obtained in step one was rapidly mixed with precursor mother liquor B obtained in step two at a volume ratio of 1:1, and a dynamic hydrogel with instantaneous gelation, pH and ROS responsiveness was obtained, denoted as BG1.

[0050] In this example, 3-aminobenzoic acid can also be replaced by 2-aminobenzoic acid or 4-aminobenzoic acid.

[0051] Example 2

[0052] The difference between this embodiment and embodiment 1 is that the water-soluble chitosan derivative for preparing the hydrogel coating precursor mother liquor B in step two is carboxymethyl chitosan, and a dynamic hydrogel with transient gelation, pH and ROS responsiveness is obtained, denoted as BG2.

[0053] Embodiment 3

[0054] The difference between this embodiment and embodiment 1 is that the water-soluble chitosan derivative for preparing the hydrogel coating precursor mother liquor B in step two is chitosan quaternary ammonium salt, and a dynamic hydrogel with transient gelation, pH and ROS responsiveness is obtained, denoted as BG3.

[0055] Embodiment 4

[0056] The difference between this embodiment and embodiment 1 is that the volume ratio of the precursor mother liquor A to the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2, and a dynamic hydrogel with transient gelation, pH and ROS responsiveness is obtained, denoted as BG4.

[0057] Embodiment 5

[0058] The difference between this embodiment and embodiment 2 is that the volume ratio of the precursor mother liquor A to the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2.

[0059] Embodiment 6

[0060] The difference between this embodiment and embodiment 3 is that the volume ratio of the precursor mother liquor A to the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2.

[0061] Embodiment 7

[0062] The difference between this embodiment and embodiment 1 is that the concentration of sodium alginate dispersed in anhydrous ethanol in step one is 0.1 g / mL, the stirring time of dispersion is 30 min, the molar ratio of added sodium periodate to sodium alginate monomer unit is 1.2:1, and the molar ratio of added ethylene glycol to sodium periodate is 1.2:1; the reaction system solution is transferred dropwise into 2 times volume of stirring anhydrous ethanol, the obtained aldehyde group concentration of oxidized sodium alginate is 8.1 mmol / g, the number average molecular weight is 120 kDa, and the molecular weight distribution DPI is 1.38.

[0063] Embodiment 8

[0064] The difference between this embodiment and embodiment 7 is that the water-soluble chitosan derivative for preparing the hydrogel coating precursor mother liquor B in step two is carboxymethyl chitosan, and a dynamic hydrogel with transient gelation, pH and ROS responsiveness is obtained.

[0065] Embodiment 9

[0066] The difference between the present example and Example 7 is that the water-soluble chitosan derivative for preparing the hydrogel coating precursor mother liquor B in step two is chitosan quaternary ammonium salt, and a dynamic hydrogel responsive to instantaneous gelation, pH and ROS is obtained.

[0067] Example 10

[0068] The difference between the present example and Example 7 is that the volume ratio of the precursor mother liquor A and the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2, and a dynamic hydrogel responsive to instantaneous gelation, pH and ROS is obtained.

[0069] Example 11

[0070] The difference between the present example and Example 8 is that the volume ratio of the precursor mother liquor A and the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2.

[0071] Example 12

[0072] The difference between the present example and Example 9 is that the volume ratio of the precursor mother liquor A and the precursor mother liquor B for preparing the hydrogel coating in step three is 1:2.

[0073] (I) FTIR characterization

[0074] The sodium alginate and oxidized sodium alginate of Example 1 of the present application were characterized by Fourier infrared spectroscopy (FTIR), and the specific process was as follows: all samples were freeze-dried and mixed with KBr dried to constant weight at a mass ratio of 1:100, ground with an agate mortar, pressed into a sheet, and then placed in a Fourier infrared spectrometer for test scanning. The scanning range was 400cm -1 ~4000cm -1 , and the results are shown in Figure 1 .

[0075] As can be seen from Figure 1 , the characteristic peaks of sodium alginate and oxidized sodium alginate at 3270~3220cm -1 , 2920cm -1 , 1594cm -1 correspond to the stretching vibration peaks of O-H, C-H and the asymmetric stretching vibration peaks of -COO - on the molecular chain, respectively, indicating that after directional oxidation with sodium periodate, the oxidized sodium alginate still retains the main functional groups such as hydroxyl and carboxyl of sodium alginate. At the same time, the hydroxyl (O-H) characteristic peak of the oxidized sodium alginate is slightly red-shifted and weakened compared with that of the sodium alginate, and the oxidized sodium alginate has a characteristic peak at 1730cm -1The new characteristic absorption peak appeared at 1710 cm-1 corresponds to the stretching vibration of aldehyde group (C=O), which indicates that oxidized sodium alginate has less hydroxyl group and new aldehyde group than sodium alginate, and also conforms to the reaction mechanism that the vicinal diol of sodium alginate is converted into aldehyde group after directional oxidation. In addition, the characteristic absorption peak of sodium alginate at 880 cm-1 corresponds to the stretching vibration of C-O-C of glycosidic bond, which becomes unobvious in oxidized sodium alginate, which may be caused by the partial breakage of sodium alginate glycosidic bond due to non-specific oxidative cleavage under the action of NaIO4 and the characteristic peak of hemiacetal structure formed by the condensation reaction of aldehyde group and part of vicinal dihydroxyl group. In summary, the comparison of the FTIR test results of sodium alginate and oxidized sodium alginate shows that part of the hydroxyl group of sodium alginate is indeed converted into aldehyde group through directional oxidation, which confirms the successful aldehyde functionalization of oxidized sodium alginate. -1

[0076] (ii) Self-healing performance

[0077] The formed hydrogel is cut into gel fragments using a craft knife, the gel fragments are contacted with each other at the cut, after a period of time, one of the gel fragments is picked up with a forceps, and it is observed that the gel fragment does not fall off from the cut under the action of gravity, which indicates that the hydrogel has self-healing ability. It is detected that the sample fragments of the hydrogels prepared in Examples 1-4 of the present application do not fall off after being spliced together for less than 1 min, which indicates that the dynamic hydrogel prepared in the present application has good self-healing efficiency, which is beneficial to prolong the service life of the hydrogel in practical application, and has a good covering effect on the medical material matrix.

[0078] (iii) Stimulus responsiveness

[0079] The dynamic polymer network of the dynamic hydrogel based on the present application is mainly crosslinked by dynamic borate ester bonds and imine bonds, so the breakage of any dynamic covalent bond will cause partial dissociation of the gel network. 20 μL of hydrogen peroxide with a mass concentration of 2.5%-3.5% is added to the 200 μL dynamic hydrogel sample prepared in Example 2 of the present application, and after 5 h, the hydrogel becomes significantly softer than the initial state, which indicates that the active oxygen of hydrogen peroxide causes the breakage of borate ester bond sites. Then the pH of the gel is adjusted to about 1-2 using 1M hydrochloric acid solution, and the hydrogel is converted from gel state to sol state after 2 d, which indicates that the imine bond of the hydrogel is broken under the action of hydrogen peroxide and hydrochloric acid. Figure 2 ​As shown, the dynamic hydrogel of this invention can slowly dissociate its gel network until it completely disintegrates in an acidic environment. This experimental phenomenon also indicates that the main driving force for gel formation in this system may originate from imine bonds, while borate ester bonds also participate in the construction of the gel network. Therefore, the dynamic hydrogel of this invention is suitable for ROS-responsive environments, such as inflammatory environments, and for low pH-responsive environments, such as acidic environments caused by bacterial metabolism. It can be used for implantation or intervention as a surface functional coating of medical materials to control the release of anti-inflammatory, antibacterial, and other drugs, thereby enhancing the therapeutic effect of medical materials.

[0080] (iv) Rheological testing

[0081] The hydrogel samples prepared in Examples 2 and 4 of this invention were subjected to rheological tests, mainly including rheological frequency scanning and strain scanning, to examine the oscillatory mechanical properties of the dynamic hydrogel samples.

[0082] The rheological frequency scanning results of the dynamic hydrogel samples prepared in Examples 2 and 4 of this invention are as follows: Figure 3 As shown, the strain scan results of the rheological test are as follows: Figure 4 As shown. From Figure 3 It can be seen that throughout the entire testing frequency range, the storage modulus (G′) of the prepared dynamic hydrogels is consistently higher than the loss modulus (G″), indicating that these transiently formed dynamic hydrogels based on dynamic borate ester bonds and imine bonds possess good rheological stability. Meanwhile, the storage modulus of the BG4 dynamic hydrogel in Example 4 is slightly higher than that of the BG2 dynamic hydrogel, indicating that under the same aldehyde concentration, increasing the content of amine and borate groups alone does not significantly affect the elastic modulus of the dynamic hydrogel. The plateau elastic modulus of both hydrogels (BG2: 10200 Pa, BG4: 11600 Pa) is also much lower than that of most medical materials currently available. As a coating, it can effectively reduce interfacial softness, alleviate stress damage to human soft tissues caused by medical materials and devices, and provide excellent protection and therapeutic effects for patients.

[0083] The rheological strain scanning results of the dynamic hydrogels prepared in Examples 2 and 4 of this invention are as follows: Figure 4 As shown, the prepared dynamic hydrogels exhibit shear-thinning capabilities, consistent with the basic characteristics of dynamically cross-linked gels. This suggests that these dynamic hydrogels have the potential for injection and printing under shear force, offering significant advantages in processing for hydrogel coatings used in medical materials. Furthermore, the shear strain and fracture strain of the BG4 dynamic hydrogel, which only increases the content of amine and boric acid groups, are significantly lower than those of the BG2 dynamic hydrogel. This indicates that the degree of shear-thinning in these dynamic hydrogels can be controlled by adjusting the proportion of the precursor solution, providing greater controllability for the processing and application of hydrogel coatings in medical materials.

[0084] (v) Biocompatibility of hydrogel coatings

[0085] The cytotoxicity of the dynamically hydrolyzed BG1, BG2, and BG3 cells from Examples 1-3 of this invention was evaluated using mouse embryonic fibroblasts (NIH / 3T3). The specific procedure was as follows: cells were seeded in 48-well plates at a density of 2 × 10⁶ cells per well. 4 After 24 hours, cells were observed to be adherent and in good condition. Different volumes of dynamic hydrocoagulated samples were placed in 48-well plates, and 200 μL of DMEM complete medium was added to each well. The volume ratios of each dynamic hydrocoagulated sample to the medium were 15.625, 31.25, 62.5, and 125 μL / mL, respectively. Medium without dynamic hydrocoagulated samples served as a control group. After 24 and 48 hours of culture, 200 μL of medium containing 10% CCK-8 reagent was added to each well. After incubation at 37°C for 1 hour, cell viability was calculated by measuring the absorbance at 450 nm using a microplate reader. The cytotoxicity test results after 48 hours are shown below. Figure 5 As shown.

[0086] from Figure 5 It was found that the cytotoxicity of the dynamic hydrogels increased with increasing volume concentration, especially at high concentrations. The three hydrogels, BG1, BG2, and BG3, exhibited significant differences in toxicity due to their different chitosan derivatives. At a low concentration of 15.625 μL / mL, there was no significant difference in toxicity among the three hydrogels, and their cell viability was comparable to the blank control group, indicating that these dynamic hydrogels all possess good biocompatibility and can effectively improve the biocompatibility of medical materials and device substrates when used as coatings.

[0087] (vi) SEM observation

[0088] After sputtering gold onto the surface of the freeze-dried dynamic hydrogel samples, the surface morphology was observed using a scanning electron microscope (SEM).

[0089] Figure 6SEM images of the dynamic hydrogels prepared in Examples 1-3 of the present application. As can be seen from the images, the gel network pore sizes of the dynamic hydrogels BG1, BG2 and BG3 prepared in Examples 1-3 are different. It is shown that by only changing the type of chitosan derivative under the premise of the same component concentration, the internal network structure of the hydrogel can be easily regulated. The micro network structure of the gel can affect the mechanical properties of the gel, and therefore by changing the type of chitosan derivative, dynamic hydrogels with suitable mechanical properties can be prepared according to the requirements of medical materials in different application scenarios. At the same time, the rich network structure of multiple scales is also conducive to the loading of drug molecules and micro-nano materials, which provides the possibility for the dynamic hydrogel to load specific biological functional substances as a medical material, and is conducive to realizing the biological functionality of the medical material hydrogel coating.

[0090] The application of the responsive dynamic hydrogel of the present application is described in detail by Examples 13-24.

[0091] Example 13

[0092] As shown in the following steps are included in this embodiment: Figure 7

[0093] Step one, the magnesium alloy WE43 wafer (diameter x thickness Φ8mm x 3mm) is polished by 400-2000 mesh sandpaper, then cleaned with acetone, deionized water and anhydrous ethanol for 15 minutes each time, the cleaning process is repeated three times, and then dried and placed in a 0.01M NaOH solution in a reaction kettle, and then subjected to alkaline heat treatment in a 120°C oven for 12 hours, and finally cleaned with deionized water and ethanol for 5 minutes, and then dried with nitrogen and stored in vacuum for standby;

[0094] Step two, the WE43 wafer prepared in step one is used as the substrate and placed in a spin coater, 100 μL of the precursor mother liquor A prepared in step one of Example 1 of the present application is spin-coated on the surface of the WE43 wafer, and then the precursor mother liquor B obtained in step two of the present application is coated on the precursor mother liquor A, thereby constructing a dynamic hydrogel coating on the surface of the medical degradable metal material WE43, which is instantaneously gelled and responsive to pH and ROS.

[0095] The WE43 wafer in this embodiment can also be replaced by other medical material substrates derived from metal titanium, magnesium, zinc, iron and their alloys, ceramic hydroxyapatite, polypropylene, polyvinyl chloride high polymer medical materials and devices, wherein the alkaline heat treatment of the medical material substrate before coating the two precursor mother liquors is not a necessary step, and the alkaline heat treatment is only for Mg, Zn and other active metals and their alloy materials.

[0096] ​The spin coating method in the present embodiment can also be replaced by brushing, doctor blading, solution casting, dip coating, spraying, inkjet / 3D printing, etc. The basic sequence is to first coat the precursor mother liquor A, and then coat the precursor mother liquor B.

[0097] Example 14

[0098] The difference between the present embodiment and Example 13 is that the precursor mother liquor A in step one of Example 2 of the present application and the precursor mother liquor B in step two of Example 2 of the present application, and the volume ratio in step three of Example 2 are used to prepare the dynamic hydrogel coating.

[0099] Example 15

[0100] The difference between the present embodiment and Example 13 is that the precursor mother liquor A in step one of Example 3 of the present application and the precursor mother liquor B in step two of Example 3 of the present application, and the volume ratio in step three of Example 3 are used to prepare the dynamic hydrogel coating.

[0101] The SEM observation results of the freeze-dried sample of the magnesium alloy WE43 disc after alkali heat treatment and covering the dynamic hydrogel coating according to Example 15 of the present application are shown in Figure 8 and Figure 9 It can be seen from Figure 8 that after the magnesium alloy WE43 disc is subjected to the alkali heat treatment of the present embodiment, a uniform and dense micro-nano flower structure is formed on the surface of the WE43 disc; the roughness of the structure is beneficial to the formation of mechanical interlocking structure between the WE43 disc and the precursor mother liquor A, and increases the friction and binding capacity between the WE43 disc and the hydrogel coating after the formation of the hydrogel coating, so that the adhesion of the coating is more firm. It can be seen from Figure 9 that after the coating of the BG3 hydrogel of Example 3 is formed on the micro-nano structure of the WE43 disc as shown in Figure 8 , the micro-network structure of the hydrogel coating does not change obviously due to the possible bonding of the carboxyl, hydroxyl and other groups in the gel with Mg 2+ , which proves the good structural stability of the hydrogel coating, and further indicates that the dynamic hydrogel coating designed in the present application is also suitable for the surface coating of active medical materials.

[0102] (VII) Contact angle test

[0103] The contact angle test is performed on the surfaces of the magnesium alloy WE43 disc, the WE43 disc after alkali heat treatment and the WE43 disc after alkali heat treatment and covering the hydrogel coating according to Examples 13-15 of the present application, respectively, to evaluate the hydrophilicity and hydrophobicity. 5 μL of deionized water is extruded from the syringe of the contact angle measuring instrument, the needle is slowly lowered until the droplet contacts the surface of the sample to be tested, and the photograph and wetting angle of the droplet on the surface at 100 s are recorded. As shown in Figure 10As shown, the contact angle of the untreated magnesium alloy WE43 disc surface is 96°, exhibiting obvious hydrophobicity; after the WE43 disc is treated with the alkaline heat method according to Examples 13-15 of this invention, the contact angle decreases to 63°, which is particularly beneficial for the spreading of precursor mother liquor A on it; subsequently, as Figure 10 As shown, after forming a hydrogel coating on the surface treated by the alkaline-thermal method, different contact angles of the chitosan derivatives exhibit different hydrophilic and hydrophobic properties. This may be related to the hydrophobic properties after chitosan and aminophenylboronic acid form borate ester bonds. Nevertheless, the hydrophilicity of the hydrogel coating can still be adjusted by controlling the type of water-soluble chitosan. For example, the hydrogel coating constructed from BG3 has a contact angle as low as 40°, exhibiting good hydrophilicity, which has high practical value for improving the hydrophilicity and biocompatibility of medical material surfaces.

[0104] Example 16

[0105] The difference between this embodiment and embodiment 13 is that the precursor mother liquor A in step one of embodiment 4 of the present invention and the precursor mother liquor B in step two of embodiment 4 of the present invention, as well as the specific volume ratio in step three of embodiment 4, are used to prepare the dynamic hydrogel coating.

[0106] Example 17

[0107] The difference between this embodiment and embodiment 13 is that the precursor mother liquor A in step one of embodiment 5 of the present invention and the precursor mother liquor B in step two of embodiment 5 of the present invention, as well as the specific volume ratio in step three of embodiment 5, are used to prepare the dynamic hydrogel coating.

[0108] Example 18

[0109] The difference between this embodiment and embodiment 13 is that the dynamic hydrogel coating is prepared by using the precursor mother liquor A in step one of embodiment 6 of the present invention, the precursor mother liquor B in step two of embodiment 6 of the present invention, and the specific volume ratio in step three of embodiment 6.

[0110] Example 19

[0111] The difference between this embodiment and embodiment 13 is that the precursor mother liquor A in step one of embodiment 7 of the present invention and the precursor mother liquor B in step two of embodiment 7 of the present invention, as well as the specific volume ratio in step three of embodiment 7, are used to prepare the dynamic hydrogel coating.

[0112] Example 20

[0113] The difference between this embodiment and embodiment 13 is that the dynamic hydrogel coating is prepared by using the precursor mother liquor A in step one of embodiment 8 of the present invention, the precursor mother liquor B in step two of embodiment 8 of the present invention, and the specific volume ratio in step three of embodiment 8.

[0114] Example 21

[0115] This example differs from Example 13 in that the precursor mother liquor A of Example 9, Step One, and the precursor mother liquor B of Example 9, Step Two, of the present application are used, along with the ratio by volume of Example 9, Step Three, to prepare the dynamic hydrogel coating.

[0116] Example 22

[0117] This example differs from Example 13 in that the precursor mother liquor A of Example 10, Step One, and the precursor mother liquor B of Example 10, Step Two, of the present application are used, along with the ratio by volume of Example 10, Step Three, to prepare the dynamic hydrogel coating.

[0118] Example 23

[0119] This example differs from Example 13 in that the precursor mother liquor A of Example 11, Step One, and the precursor mother liquor B of Example 11, Step Two, of the present application are used, along with the ratio by volume of Example 11, Step Three, to prepare the dynamic hydrogel coating.

[0120] Example 24

[0121] This example differs from Example 13 in that the precursor mother liquor A of Example 12, Step One, and the precursor mother liquor B of Example 12, Step Two, of the present application are used, along with the ratio by volume of Example 12, Step Three, to prepare the dynamic hydrogel coating.

[0122] The above description is only the preferred embodiment of the present application, and is not intended to limit the present application. Any simple modification, change, and equivalent change of the above embodiment according to the technical essence of the present application are still within the protection scope of the technical solution of the present application.

Claims

1. A method for preparing a responsive dynamic hydrogel, characterized by, The method comprises the following steps: Step one, preparation of hydrogel precursor mother liquor A: sodium alginate is dispersed in anhydrous ethanol, and a sodium periodate aqueous solution is used for directional oxidation reaction under light shielding conditions; after the reaction is terminated, purified aldehyde group-containing oxidized sodium alginate is obtained, then the aldehyde group-containing oxidized sodium alginate is dissolved in a hot polyvinyl alcohol solution to obtain the precursor mother liquor A; Step two, preparation of hydrogel precursor mother liquor B: a water-soluble chitosan derivative is dissolved in deionized water, then a small molecule of aminophenylboronic acid is added, and heated and stirred to react to obtain the precursor mother liquor B; Step three, preparation of the hydrogel: the precursor mother liquor A obtained in step one and the precursor mother liquor B obtained in step two are rapidly stirred and mixed uniformly at a volume ratio of 1:1-2 to obtain a dynamic hydrogel which is instantaneously gelled and pH and ROS responsive.

2. The method of claim 1, wherein the responsive dynamic hydrogel is prepared by the steps of: The concentration of the sodium alginate dispersed in the anhydrous ethanol in step one is 0.1 g / mL-0.2 g / mL.

3. The method for preparing a responsive dynamic hydrogel according to claim 1, characterized in that, The molar ratio of sodium periodate to sodium alginate monomer units in the sodium periodate aqueous solution in step one is 0.5:1-1.2:

1.

4. The method of claim 1, wherein the responsive dynamic hydrogel is prepared by the steps of: The method for terminating the reaction in step one is to add ethylene glycol, and the molar ratio of ethylene glycol to sodium periodate in the sodium periodate aqueous solution is 1.5:1-1.2:

1.

5. The method for preparing a responsive dynamic hydrogel according to claim 1, characterized in that, The method for purification in step one is: the reaction system solution is transferred drop by drop to 2-3 times of stirring anhydrous ethanol, then the precipitate is collected by filtration, the precipitate is dissolved in deionized water and dialyzed in deionized water, and then freeze-dried.

6. The method of claim 1, wherein the responsive dynamic hydrogel is prepared by the steps of: The concentration of the total polymer in the precursor mother liquor A in step one includes aldehyde group-containing oxidized sodium alginate and polyvinyl alcohol, which is 0.14 g / mL, the concentration of polyvinyl alcohol is 0.1 g / mL, and the mass ratio of aldehyde group-containing oxidized sodium alginate to polyvinyl alcohol is 5:

2.

7. The method for preparing a responsive dynamic hydrogel according to claim 1, characterized in that, The water-soluble chitosan derivative in step two is carboxymethyl chitosan, carboxylated chitosan, chitosan quaternary ammonium salt, chitosan hydrochloride or chitosan lactate, which is dissolved in deionized water at a concentration of 0.02 g / mL; the small molecule of aminophenylboronic acid is 2-amino phenylboronic acid, 3-amino phenylboronic acid or 4-amino phenylboronic acid, which is added at a concentration of 0.018 g / mL.

8. Use of a responsive dynamic hydrogel prepared according to the method of any one of claims 1 to 7, characterized in that, The precursor mother liquor A obtained in step one is coated on the medical material substrate, then the precursor mother liquor B obtained in step two is coated on the precursor mother liquor A, and a dynamic hydrogel coating which is instantaneously gelled and pH and ROS responsive is obtained on the surface of the medical material.

9. Use according to claim 8, characterized in that, The coating method of the precursor mother liquor A and the precursor mother liquor B is derived from brushing, scraping, solution casting, dipping, spinning, spraying, inkjet / 3D printing.

10. Use according to claim 8, characterized in that, The medical material substrate is derived from metal titanium, magnesium, zinc, iron and their alloys, ceramic hydroxyapatite, polypropylene, polyvinyl chloride high polymer medical materials and devices.

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