Preparation method and application of ultrasonic-excited multifunctional hydrogel

Abstract

The invention discloses a preparation method and application of ultrasonic-excited multifunctional hydrogel, and belongs to the technical field of biomedical engineering. The multifunctional hydrogel is prepared from an injectable hydrogel carrier and reduced molybdate loaded on the injectable hydrogel carrier, the injectable hydrogel carrier is prepared from aminated hyaluronic acid and aldehyde hyaluronic acid through a Schiff base covalent cross-linking reaction. The multifunctional hydrogel can achieve different effects through two times of ultrasonic treatment. After the first ultrasonic treatment, the reduced molybdate is quickly converted into oxidized molybdate, and active oxygen in the solution is quickly removed to generate oxygen, so that the anti-oxidation effect is realized; further, under ultrasonic treatment, the oxidized molybdate shows a glucose oxidase-like effect and generates singlet oxygen, so that the effects of reducing blood sugar and sterilizing are realized. The multifunctional hydrogel can simultaneously achieve the effects of resisting oxidation, reducing blood glucose and sterilizing under ultrasound, and can be used for ultrasonic assisted treatment of diabetic wounds to promote recovery of the wounds of diabetic patients.

Description

Technical Field

[0001] The invention relates to the technical field of biomedical engineering, and in particular to a preparation method and application of an ultrasound-stimulated multifunctional hydrogel. Background Art

[0002] Diabetes is a metabolic disease characterized by high blood sugar. Long-term high blood sugar can cause various complications, especially diabetic ulcers caused by difficulty in wound healing. These ulcers can lead to a series of serious problems, such as reduced quality of life, severe infection and even amputation. Therefore, the healing of chronic diabetic wounds remains one of the major clinical challenges.

[0003] More and more studies have shown that the main challenges to overcoming diabetic wound healing include high blood sugar levels, persistent inflammatory responses, excessive oxidative stress, and external bacterial infection. Continuous inflammation causes excessive oxidative stress in the wound microenvironment and accumulates a large amount of reactive oxygen species (ROS). Excessive ROS not only damages tissues, inhibits wound healing, but also prompts macrophages to transform into M1 macrophages, leading to the secretion of pro-inflammatory inducing factors such as tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6). At the same time, high blood sugar can cause the phagocytic ability of immune cells such as macrophages and neutrophils to decrease, resulting in a decrease in the body's anti-inflammatory ability. In addition, the high blood sugar level in diabetic wounds greatly increases the chance of bacterial infection of the wound, and bacterial infection will further aggravate the accumulation of ROS and the hypoxic environment of the wound. However, the current clinical treatment of diabetic wounds mainly focuses on regulating blood sugar, inhibiting inflammation, keeping the wound clean, and promoting tissue proliferation. Although these methods guarantee wound healing to a certain extent, there are cumbersome steps and high costs in implementation. Therefore, developing a simple and efficient multifunctional material that can integrate functions such as eliminating ROS, lowering blood sugar, and antibacterial on the same substrate has become an important clinical need. Summary of the invention

[0004] The purpose of the present invention is to provide a preparation method and application of an ultrasound-stimulated multifunctional hydrogel to solve the problem in the prior art that diabetic wounds are difficult to heal due to factors such as persistent inflammation, hyperglycemia and bacterial infection in the microenvironment. The multifunctional hydrogel of the present invention can achieve anti-inflammatory, hypoglycemic and antibacterial effects under ultrasound, thereby effectively promoting wound healing.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention is: an ultrasound-stimulated multifunctional hydrogel, which is composed of an injectable hydrogel carrier and reduced molybdate loaded on the injectable hydrogel carrier; the injectable hydrogel carrier is obtained by a Schiff base covalent cross-linking reaction of amino hyaluronic acid and aldehyde hyaluronic acid.

[0007] Furthermore, the loading concentration of the reduced molybdate in the injectable hyaluronic acid hydrogel is 0-1000 μg / ml, and is not 0; preferably 0-500 μg / ml, and is not 0.

[0008] Furthermore, the preparation method of the reduced molybdate comprises: mixing an aqueous molybdate solution and a disodium hydrogen phosphate aqueous solution to obtain a mixed solution; adding L-ascorbic acid to the mixed solution to carry out a reduction reaction; and after the reaction is completed, sequentially subjecting the mixed solution to ethanol sedimentation, centrifugation, washing, dialysis and freeze-drying to obtain the reduced molybdate.

[0009] Furthermore, the molybdate is ammonium heptamolybdate; the molar ratio of molybdate to disodium hydrogen phosphate in the mixed solution is 1:0.6-2.5; and the molar ratio of molybdate contained in the mixed solution to the added L-ascorbic acid is 1:0.1-3.6.

[0010] Furthermore, the molybdate aqueous solution is prepared by dissolving ammonium heptamolybdate in ultrapure water at a solid-liquid ratio of 1-5 g:2-5 ml to obtain the molybdate aqueous solution.

[0011] Furthermore, the L-ascorbic acid is added in the form of a solution, and the concentration of the L-ascorbic acid solution is 50-600 mg / ml.

[0012] The reduced molybdate prepared by the above method is a Keggin-type molybdate with a certain degree of reduction (containing Mo 6+ and Mo 5+ , L-ascorbic acid converts the Mo in ammonium heptamolybdate 6+ Partially reduced to Mo 5+ ); the Keggin-type molybdate having a certain degree of reduction (wherein Mo 5+ The reduced molybdate can be converted into the oxidized molybdate (molybdenum in the form of Mo) under ultrasound for 3-8 minutes. 6+ exists in the form of ).

[0013] The above-mentioned Keggin-type molybdate with a certain degree of reduction has an adaptive electronic structure, in which the molybdenum ions can undergo a valence change under the action of ultrasound, which can be 5+ to Mo 6+ The conversion of ROS into O2 can help macrophages transform into M2 anti-inflammatory type and promote wound healing.

[0014] The second technical solution of the present invention: The preparation method of the above-mentioned ultrasonically excited multifunctional hydrogel comprises the following steps: dissolving reduced molybdate, amino hyaluronic acid and aldehyde hyaluronic acid in water respectively to obtain reduced molybdate aqueous solution, amino hyaluronic acid aqueous solution and aldehyde hyaluronic acid aqueous solution; mixing the reduced molybdate aqueous solution with the amino hyaluronic acid aqueous solution to obtain an amino hyaluronic acid aqueous solution containing reduced molybdate; mixing the amino hyaluronic acid aqueous solution containing reduced molybdate with the aldehyde hyaluronic acid aqueous solution, and carrying out Schiff base covalent cross-linking reaction to obtain the ultrasonically excited multifunctional hydrogel.

[0015] The Schiff base covalent cross-linking reaction is carried out at room temperature and is generally completed within 30 seconds to 5 minutes.

[0016] During the preparation process, formaldehyde-modified hyaluronic acid and amino-modified hyaluronic acid form dynamic bonds through Schiff base covalent cross-linking reaction. The broken Schiff base bonds can be reformed to repair the structure of the material, thus having self-healing properties.

[0017] The reversibility of the Schiff base dynamic bond allows the hydrogel to temporarily break some bonds under shear stress (such as when passing through a syringe), thereby reducing viscosity. After injection, due to the disappearance of stress, the Schiff base bond can be formed again, allowing the hydrogel to re-condense at the target site. Therefore, this characteristic of the Schiff base dynamic bond allows the hydrogel to maintain fluidity during injection and quickly gel after injection.

[0018] Furthermore, the concentration of the reduced molybdate aqueous solution is 0-10 mg / ml and is not 0; the volume ratio of the reduced molybdate aqueous solution to the amino hyaluronic acid aqueous solution is 1:2-7; the volume ratio of the amino hyaluronic acid aqueous solution containing reduced molybdate to the aldehyde hyaluronic acid aqueous solution is 0.8-2.5:1.

[0019] Furthermore, the concentration of the amino hyaluronic acid aqueous solution is 20 mg / ml; the concentration of the aldehyde hyaluronic acid aqueous solution is 50 mg / ml.

[0020] Furthermore, after the amino-modified hyaluronic acid aqueous solution containing reduced molybdate is mixed with the aldehyde-modified hyaluronic acid aqueous solution, the concentration of reduced molybdate in the mixed system is 0-1000 μg / ml, and is not 0.

[0021] Furthermore, the preparation method of the amino hyaluronic acid comprises: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 4.8-9.5 times the molar number of the hyaluronic acid repeating unit to the hyaluronic acid aqueous solution, adjusting the pH value of the mixed system to 6.0-7.0, stirring and reacting at room temperature for 10-20 hours, and then dialyzing and freeze-drying in sequence to finally obtain the amino hyaluronic acid. This preparation process is essentially a carboxyl-amino coupling reaction modification process of hyaluronic acid.

[0022] Furthermore, the preparation method of the aldehyde-modified hyaluronic acid comprises: adding sodium periodate in a molar amount of 0.6-1.5 times the molar number of hyaluronic acid repeating units to a hyaluronic acid aqueous solution, stirring and dissolving at room temperature, and then placing in a dark environment and stirring for 4-8 hours, and then quenching the reaction with excess ethylene glycol, and after the reaction is completed, dialyzing and freeze-drying are performed in sequence to finally obtain the aldehyde-modified hyaluronic acid. This preparation process is essentially an oxidative modification process of hyaluronic acid.

[0023] Furthermore, the mass percentage concentration of the hyaluronic acid aqueous solution is 0.8-2.5%, and the molecular weight (number average molecular weight) of the hyaluronic acid therein is 100,000-800,000.

[0024] Furthermore, the number of moles of hyaluronic acid repeating units can be calculated based on the mass of hyaluronic acid and the molecular weight of the repeating unit (monomer molecular weight). Number of moles of hyaluronic acid repeating units = mass of hyaluronic acid / molecular weight of monomer.

[0025] Furthermore, in the process of preparing reduced molybdate, amino hyaluronic acid and aldehyde hyaluronic acid, the dialysis is performed with ultrapure water for three days, and the molecular weight cutoff of the dialysis bag used is 3500Da; the freeze-drying is performed by quick freezing with liquid nitrogen and then keeping it in a vacuum freeze dryer for 24-72 hours until the sample is dry.

[0026] The ultrasound-stimulated multifunctional hydrogel prepared by the above method has injectability, self-healing and skin adhesion; and the injectable hyaluronic acid hydrogel carrier is a porous structure, and the reduced molybdate is evenly dispersed in the pores and on the surface of the hydrogel. The above ultrasound-stimulated multifunctional hydrogel has the effects of eliminating accumulated ROS, generating O2, reducing blood sugar in the wound microenvironment and antibacterial under ultrasound, and has a good effect on promoting the recovery of diabetic infected wounds.

[0027] The third technical solution of the present invention: Application of the above-mentioned ultrasound-stimulated multifunctional hydrogel in the preparation of dressings for treating chronic inflammatory wounds.

[0028] Furthermore, the chronic inflammatory wound is a chronic inflammatory wound of a diabetic patient.

[0029] Furthermore, the ultrasound-excited multifunctional hydrogel is injected into the wound, and the multifunctional hydrogel at the wound is subjected to ultrasound treatment; the ultrasound treatment is performed twice, each ultrasound treatment lasts for 3-8 minutes, and the interval between the two ultrasound treatments is 3-5 minutes.

[0030] Furthermore, the ultrasonic parameters include: frequency of 0.5-2.0 MHz, duty cycle of 50%, power of 0.5-1.2 W / cm 2 .

[0031] In order to improve the persistent inflammatory environment of hyperglycemic wounds and the problems of decreased immune cell function and susceptibility to infection caused by high sugar, the inventors conducted a series of studies and found that ultrasound can not only promote the conversion of reduced molybdate to oxidized molybdate (Mo 5+ →Mo 6+ ), and the oxidized molybdate has a glucose oxidase-like effect under ultrasound, and the effect of reacting with hydrogen peroxide to produce singlet oxygen. Based on this, the present invention designs and synthesizes a reduced molybdate (Keggin-type molybdate with a certain degree of reduction), so that it can achieve a valence state change under short-term ultrasound, and then uses a modified hyaluronic acid injectable hydrogel as a carrier to prepare an ultrasound-excited multifunctional hydrogel. The hydrogel can achieve different effects through two stages of ultrasound. First, the first stage of ultrasound promotes the rapid conversion of reduced molybdate in Keggin-type molybdate with a certain degree of reduction into oxidized molybdate, and in this process, ROS is quickly removed to produce O2, which improves persistent inflammation and achieves an antioxidant effect; the oxidized molybdate has a glucose oxidase-like effect and produces singlet oxygen under ultrasound in the second stage, achieving hypoglycemic and bactericidal effects. Moreover, the modified multifunctional hydrogel has good biosafety and can be directly injected into the irregular wound surface of diabetic wounds.

[0032] The present invention discloses the following technical effects:

[0033] (1) The multifunctional hydrogel of the present invention has the function of protecting wounds from external stimulation and infection, and has injectability and self-healing properties, and can be used for wounds with complex shapes and various depths.

[0034] (2) The multifunctional hydrogel of the present invention can achieve oxidation of molybdate under the first short-term ultrasound by loading reduced molybdate, and in the process eliminate excess ROS, generate O2, and improve the persistent severity of the wound and the hypoxic environment.

[0035] (3) The oxidized molybdate converted from the reduced molybdate loaded on the multifunctional hydrogel of the present invention after the first ultrasound can achieve a glucose oxidase-like effect under a short-time ultrasound action (second ultrasound), improve the hyperglycemic environment of the wound microenvironment, and produce a small amount of hydrogen peroxide.

[0036] (4) The multifunctional hydrogel of the present invention, after the second ultrasound, produces hydrogen peroxide which can react with oxidized molybdate to generate singlet oxygen for killing bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0038] Figure 1 The performance characteristics of POM with different reduction degrees prepared in Example 1, wherein A is the UV-visible absorption spectrum of POM with different reduction degrees; B is the proportion of oxidized molybdate after ultrasound; C and D are the ROS elimination effects of POM with different reduction degrees under non-ultrasound (C) and ultrasound (D) tested by DCFH, respectively; E is a quantitative bar graph of the remaining ROS; F is the POM with different concentrations re5 ROS elimination effect; +US represents ultrasonic treatment, and -US represents non-ultrasonic treatment (the +US and -US in the following figures all represent this meaning).

[0039] Figure 2 This is a diagram of the process of preparing a multifunctional hydrogel by mixing an amino-containing hyaluronic acid aqueous solution and an aldehyde-containing hyaluronic acid aqueous solution containing POM in Example 3.

[0040] Figure 3 This is the SEM image of the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3.

[0041] Figure 4 This is the EDS image of the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3;

[0042] Figure 5 Fourier transform infrared spectra of HA-NH2, HA-CHO prepared in Example 2 and HA prepared in Example 3.

[0043] Figure 6 The rheological analysis results of the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3, wherein A is the change in storage modulus; and B is the change in loss modulus.

[0044] Figure 7 This is a verification of the adhesion of the hydrogel (the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3) to the skin.

[0045] Figure 8 The image is a verification image of the injectability of the hydrogel (the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3).

[0046] Fig. 9 This is a verification of the repairability of the hydrogel (the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3).

[0047] Fig.10 The figures are the ROS elimination efficiency of the hydrogel loaded with different concentrations of POM prepared in Example 3 under ultrasonic and non-ultrasonic conditions, wherein A is a schematic diagram of ultrasound-enhanced POM hydrogel elimination of ROS and conversion into O2, and B is the elimination efficiency of H2O2 by the hydrogel after ultrasonic treatment; C and D are the X-ray photoelectron spectroscopy (XPS) of the hydrogel loaded with 500 μg / ml POM before and after ultrasound, respectively, E is the ROS elimination rate of the hydrogel loaded with different concentrations of POM after ultrasonic treatment or standing at room temperature, and F is the ROS elimination efficiency of the hydrogel loaded with 500 μg / ml POM after ultrasonic treatment for different times or standing at room temperature for different times.

[0048] Fig.11 The oxygen production effect of the hydrogel loaded with different concentrations of POM prepared in Example 3 under ultrasonic and non-ultrasonic conditions, wherein A is the oxygen production under different ultrasonic treatment times; B is the oxygen production under different room temperature standing treatment times; C is the oxygen production when treated for 8 minutes under ultrasonic and non-ultrasonic conditions.

[0049] Fig.12 The hypoglycemic effect of the hydrogel loaded with different concentrations of POM prepared in Example 3 under ultrasonic and non-ultrasonic conditions, wherein A is the UV-visible absorption spectrum after ultrasonic treatment; B is the UV-visible absorption spectrum without ultrasonic treatment; C is the comparison of the maximum absorbance of TMB after ultrasonic treatment and without ultrasonic treatment.

[0050] Fig.13 The singlet oxygen production effect of the hydrogel loaded with different concentrations of POM prepared in Example 3 under ultrasonic and non-ultrasonic conditions, wherein A is the fluorescence emission spectrum after ultrasonic treatment; B is the fluorescence emission spectrum without ultrasonic treatment; C is the ratio change of the maximum fluorescence intensity of SOSG at 521 nm.

[0051] Fig.14These are the cytotoxicity test results of the hydrogels loaded with different concentrations of POM prepared in Example 3, wherein A is the cell activity of RAW264.7 cells under different conditions; and B is the cell activity of L929 cells under different conditions.

[0052] Fig.15 The results of antibacterial activity verification of the hydrogel, wherein A is the bactericidal effect of different groups (PBS+US, HA+US, HP-US, HP+US) on Staphylococcus aureus verified by plate counting method and crystal violet staining method; B is a quantitative analysis bar graph of plate counting method and crystal violet staining method of different groups on Staphylococcus aureus; C is the bactericidal effect of different groups (PBS+US, HA+US, HP-US, HP+US) on Escherichia coli verified by plate counting method and crystal violet staining method; D is a quantitative analysis bar graph of plate counting method and crystal violet staining method of different groups on Escherichia coli;

[0053] Fig.16 These are the results of animal experiments verifying that hydrogels promote wound recovery, where A is a representative digital photo of wound healing in different groups (PBS+US, HA+US, POM+US, HP-US, HP+US) on different days; B is a schematic diagram of wound healing; C is the wound healing rate of each group on the 7th and 14th days; D is the blood sugar change curve on different days; E is the colony analysis of the wound surface of mice after the treatment was completed.

[0054] Fig.17 The present invention relates to a specific embodiment of the present invention, wherein a is a schematic diagram of the preparation process of the ultrasound-stimulated multifunctional hydrogel and a schematic diagram of the effect principle of the ultrasound-stimulated multifunctional hydrogel, wherein b is a schematic diagram of the principle of the ultrasound-stimulated multifunctional hydrogel eliminating ROS and converting it into O2, and c is a schematic diagram of the ultrasound-stimulated multifunctional hydrogel exerting antioxidant, hypoglycemic and bactericidal effects at the wound to promote wound healing. DETAILED DESCRIPTION

[0055] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0056] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0057] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0058] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0059] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0060] Description of terms or abbreviations involved in the specific implementation of the present invention:

[0061] POM refers to reduced molybdate (Keggin-type molybdate with a certain degree of reduction);

[0062] HA-NH2 refers to amino-functionalized hyaluronic acid;

[0063] HA-CHO refers to aldehyde-functionalized hyaluronic acid;

[0064] HA refers to an injectable hydrogel formed by amino-functionalized hyaluronic acid and aldehyde-functionalized hyaluronic acid without POM loading;

[0065] HP refers to an injectable hydrogel loaded with POM formed by amino-functionalized hyaluronic acid and aldehyde-functionalized hyaluronic acid, i.e., the ultrasound-stimulated multifunctional hydrogel of the present invention;

[0066] ROS is the general term for reactive oxygen species. Common ROS are H2O2, 1 O2、O2 - ;

[0067] The Gox-like effect refers to the glucose oxidase-like effect, which can decompose glucose into hydrogen peroxide and gluconic acid.

[0068] The room temperature or normal temperature involved in the specific implementation manner of the present invention specifically refers to 20-25°C.

[0069] The schematic diagram of the preparation process of the ultrasound-stimulated multifunctional hydrogel involved in the specific embodiment of the present invention and the schematic diagram of the effect principle of the ultrasound-stimulated multifunctional hydrogel are as follows: Fig.17 As shown, a is a schematic diagram of the preparation process of ultrasound-stimulated multifunctional hydrogel, b is a schematic diagram of the principle of ultrasound-stimulated multifunctional hydrogel eliminating ROS and converting it into O2, and c is a schematic diagram of ultrasound-stimulated multifunctional hydrogel exerting antioxidant, hypoglycemic and bactericidal effects at the wound to promote wound healing.

[0070] The molecular weight of the hyaluronic acid used in the following examples and comparative examples is 100,000.

[0071] Example 1

[0072] Preparation of POM

[0073] 2 g (1.72 mmol) of ammonium heptamolybdate was dissolved in 3 ml of ultrapure water to obtain an ammonium heptamolybdate aqueous solution, to which 1 ml of a 150 mg / ml (containing 1.25 mmol of sodium dihydrogen phosphate) aqueous solution of sodium hydrogen phosphate was added, and the mixture was stirred at room temperature for uniform dissolution to obtain a mixed solution; the mixed solution was divided into 6 equal parts, and 0.5 ml of 50, 100, 150, 200, 250, and 300 mg / ml of L-ascorbic acid solution (containing 0.14, 0.28, 0.42, 0.56, 0.7, and 0.84 mmol of L-ascorbic acid, respectively) were added, and the reduction reaction was carried out at room temperature for 2 h. After the reaction was completed, 15 ml of anhydrous ethanol was added to each of them for sedimentation, and then centrifuged at 8000 rpm for 15 min. Then, the mixture was washed with anhydrous ethanol and pure water for 3 times, and then dialyzed with a dialysis bag with a molecular weight cutoff of 3500 Da for 48 hours. Finally, it was freeze-dried for 48 hours (after quick freezing with liquid nitrogen, it was kept in a vacuum freeze dryer for 48 hours) to obtain POM with different reduction degrees. The more L-ascorbic acid was added, the higher the reduction degree of the obtained POM was. According to the amount of L-ascorbic acid added, they were named POM according to the reduction degree from low to high. re1 、POM re2 、POM re3 、POM re4 、POM re5 、POM re6 .

[0074] Example 2

[0075] Preparation of functionalized hyaluronic acid

[0076] (1) Preparation of aldehyde-modified hyaluronic acid: 100 ml of 1.5 wt% hyaluronic acid aqueous solution (100 ml of water + 1.5 g of hyaluronic acid) was prepared, and 750 mg (3.5 mmol) of sodium periodate was added thereto. After stirring and dissolving at room temperature, the mixture was placed in a dark environment and stirred for 8 h. After the reaction was complete, an excess of ethylene glycol was used to quench the reaction. After the reaction was completed, the mixture was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 3500 Da and then freeze-dried for 2 days to obtain aldehyde-modified hyaluronic acid.

[0077] (2) Preparation of amino hyaluronic acid: Prepare 100 ml of 2.5 wt% aqueous solution of hyaluronic acid, add 2.7 g (17.4 mmol) of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 5.2 g (38.5 mmol) of 1-hydroxybenzotriazole to activate the carboxyl group, then adjust the pH value of the mixed system to 6.5, stir and react at room temperature for 12 h. After the reaction, dialyze with a dialysis bag with a molecular weight cutoff of 3500 Da for 3 days and then freeze-dry for 2 days to obtain amino hyaluronic acid.

[0078] Example 3

[0079] Preparation of multifunctional hydrogels loaded with different concentrations of POM

[0080] The amino hyaluronic acid and aldehyde hyaluronic acid prepared in Example 2 were dissolved in ultrapure water to obtain an aqueous solution of amino hyaluronic acid and an aqueous solution of aldehyde hyaluronic acid with a concentration of 20 mg / ml and 50 mg / ml, respectively; the POM prepared in Example 1 was re5Dissolved in ultrapure water to obtain 6 portions of POM aqueous solutions with different concentrations, 0, 1, 2, 3, 5, 10 mg / ml respectively; 6 portions of 250 μL of POM aqueous solutions with different concentrations were mixed with the above-mentioned amino hyaluronic acid aqueous solution at a volume ratio of 1:5 (250 μL POM aqueous solution: 1.25ml amino hyaluronic acid aqueous solution) are mixed to obtain 6 amino hyaluronic acid aqueous solutions containing POM with different concentrations (the volume is 1.5ml), and then the 6 amino hyaluronic acid aqueous solutions containing POM with different concentrations are mixed with the above-mentioned aldehyde hyaluronic acid aqueous solution according to a volume ratio of 1.5:1 (1.5ml amino hyaluronic acid aqueous solution containing POM with different concentrations: 1ml aldehyde hyaluronic acid aqueous solution). The uniformly mixed solution can undergo Schiff base covalent cross-linking reaction in a short time (within 3min) to form an injectable hydrogel, and 6 ultrasonically excitable multifunctional hydrogels (including 0μg / ml loaded with POM) with different concentrations (0, 100, 200, 300, 500, 1000μg / ml, where the loading concentration refers to the loading concentration of POM in the uniformly mixed solution, that is, the mass of POM / the sum of the volumes of the amino hyaluronic acid aqueous solution, the aldehyde hyaluronic acid aqueous solution and the POM aqueous solution) can be obtained. The product of POM is actually injectable hyaluronic acid hydrogel HA without POM loading, and the product loaded with 100, 200, 300, 500, 1000 μg / ml POM is HP).

[0081] Test Example 1

[0082] Screening POM with appropriate reduction degree

[0083] In order to achieve the conversion of POM into oxidized POM in a short time and still have good ROS elimination performance in the process, this test example screened POM with different reduction degrees prepared in Example 1, and by verifying the content of oxidized POM and ROS elimination performance after ultrasound, the POM with the most suitable reduction degree was screened for the next experiment.

[0084] The various properties of POM with different reduction degrees prepared in Example 1 are characterized as follows Figure 1 As shown in the figure, the UV-visible absorption spectra of POM with different reduction degrees were first measured (such as Figure 1 As shown in A in the figure, it can be seen that the absorbance of POM at 865nm gradually increases with the increase of reduction degree. The characteristic absorption curve of POM can be used to quantify the reduction degree. Then, POM with different reduction degrees was dissolved in ultrapure water to obtain POM aqueous solutions with the same concentration and different reduction degrees. The POM solutions with the same concentration (2 mg / ml) and different reduction degrees were ultrasonically treated for 5 min (frequency 1.0 MHz, duty cycle 50%, power 1.0 W / cm 2) and then tested its absorbance at 865 nm, and calculated the proportion of oxidized molybdate after ultrasound according to the ratio of absorbance at 865 nm before and after ultrasound (e.g. Figure 1 As shown in B), you can see POM re1 、POM re2 Almost completely oxidized, POM re3 、POM re4 、POM re5 The oxidation rates of POM re6 The oxidation rate is low. Then the ROS elimination performance of POM with different reduction degrees was verified. Specifically, DCFH was used to explore the ROS elimination performance of POM with different reduction degrees under non-ultrasound and ultrasound (frequency 1.0 MHz, duty cycle 50%, power 1.0 W / cm 2 , ultrasound time 5min) of ROS elimination effect, the specific operation is: 3ml 10mmol / L potassium superoxide solution as ROS source, POM with different reduction degrees is mixed with it (the concentration of POM in the mixed system is 500μg / ml), then ultrasonic or static treatment is performed, and 1μL DCFH probe is added for fluorescence intensity detection (the results are shown in Figure 1 As shown in C and D in the figure, C is under non-ultrasound and D is under ultrasound), and a quantitative bar graph of the remaining ROS is drawn (as shown in Figure 1 As shown in E), it can be seen that POM re5 More than 75% of ROS have been eliminated, compared to POM re1 -POM re4 The ROS elimination rate of POM is not outstanding. re6 Although the elimination rate of POM is higher, its high oxidation rate will affect the subsequent Gox-like effect and the production of singlet oxygen. re5 For subsequent experiments, this test case also explored different concentrations of POM re5 The ROS elimination efficiency (such as Figure 1 As shown in F, Control represents the blank control, and 0.3 mg / ml represents POM in the mixed system. re5 The concentration of POM in the mixed system is 0.3mg / ml, and 0.5mg / ml represents re5 The concentration is 0.5 mg / ml), which shows that ultrasound can greatly enhance the ROS elimination efficiency of POM and reduce the concentration dependence of POM.

[0085] Test Example 2

[0086] Surface properties characterization of multifunctional hydrogels

[0087] The process of preparing a multifunctional hydrogel by mixing an aqueous solution of amino-modified hyaluronic acid containing POM and an aqueous solution of aldehyde-modified hyaluronic acid in Example 3 is shown in FIG. Figure 2 The microstructure of the hydrogel was observed by scanning electron microscopy, where the SEM image of the multifunctional hydrogel loaded with 500 μg / ml POM is shown in Figure 3 As shown in the figure, it can be seen that the hydrogel has a microporous structure. Further local magnification shows that POM is evenly dispersed on the hydrogel. Through the energy electron scattering analysis of the Mo element (EDS figure as shown in Figure 4 As shown), the uniform distribution of Mo element can also be proved.

[0088] Test Example 3

[0089] Functional characterization of hyaluronic acid

[0090] Fourier transform infrared spectroscopy analysis was performed on HA-NH2, HA-CHO prepared in Example 2 and HA prepared in Example 3. Appropriate amounts of HA, HA-NH2, and HA-CHO were respectively put into an agate mortar with a certain amount of potassium bromide, ground and mixed evenly, and the mixture was pressed into a tablet and placed in an infrared spectrometer at 4000 cm -1 -400cm -1 The Fourier transform infrared spectrum is tested within the scanning range. Figure 5 As shown in Figure 1, the characteristic chemical structures of HA, HA-NH2, and HA-CHO were determined by analyzing the peak wavenumber positions in the spectra. The Fourier transform infrared spectrum showed that HA-NH2 (1720 cm -1 ) and HA-CHO (2820cm -1 ) characteristic peaks, indicating that the surface hyaluronic acid has been modified successfully.

[0091] Test Example 4

[0092] Characterization of injectability, viscosity, and self-healing properties of multifunctional hydrogels

[0093] (1) Characterization of injectability and viscosity: First, a rotational rheometer was used to perform a time scan on the multifunctional hydrogel (HP) loaded with 500 μg / ml POM in Example 3. The rheological analysis results are shown in Figure 2. Figure 6 As shown in Figure 2, the changes in the storage modulus (A) and loss modulus (B) of the hydrogel confirm the gel-forming property and injectability of the hydrogel. The hydrogel is adhered to the skin surface, such as Figure 7 As shown in the figure, the good viscosity of HP hydrogel is demonstrated by changing the angles. HP hydrogel is sucked into a 1 ml syringe and the syringe plunger is manually pushed, as shown in the figure. Figure 8As shown, the hydrogel can be easily squeezed out and write the letters "SIFE" in a petri dish. The injectability of the hydrogel allows for precise coverage of the wound, which can improve the seal on the wound and reduce the risk of infection.

[0094] (2) Self-healing property: Hydrogels with self-healing properties can also repair themselves after being damaged, which greatly extends the service life of the hydrogel. Fig. 9 As shown, a piece of hydrogel (the multifunctional hydrogel loaded with 500 μg / ml POM in Example 3) was divided into two pieces of hydrogel of the same size from the middle, one of which was dyed purple-red with rhodamine B, and the other kept its original light blue color (first dyed and then cut into two pieces from the middle with scissors). The two hydrogels were reassembled and placed in a culture dish. After 5 minutes, they were lifted up with tweezers. The two separated hydrogels adhered to each other and did not break even after a certain pulling force was applied to both ends.

[0095] Test Example 5

[0096] Characterization of ROS elimination performance of multifunctional hydrogels

[0097] The hydrogels loaded with different concentrations of POM (0, 100, 200, 300, 500 μg / ml) prepared in Example 3 were placed in 10 mmol / L potassium superoxide solution using 3 ml of 10 mmol / L potassium superoxide solution as the ROS source. After 5 minutes of ultrasonic treatment (frequency 1.0 MHz, duty cycle 50%, power 1.0 W / cm 2 ) or at room temperature for 5 minutes, and then 2',7'-dichlorofluorescein (DCFH, added in an amount of 1 μL) was used to quantitatively observe the ROS elimination efficiency of the HP hydrogel. Fig.10 As shown, A is a schematic diagram of ultrasound-enhanced POM hydrogel elimination of ROS and conversion to O2, B is the efficiency of hydrogel elimination of H2O2 after ultrasound treatment; C and D are X-ray photoelectron spectroscopy (XPS) of hydrogels loaded with 500μg / ml POM before and after ultrasound, E is the ROS elimination rate of hydrogels loaded with different concentrations of POM after ultrasound treatment or standing at room temperature, and F is the ROS elimination efficiency of hydrogels loaded with 500μg / ml POM after different ultrasound treatment times or standing at room temperature for different times. Fig.10 It can be seen that under ultrasonic treatment, the ROS elimination efficiency of HP hydrogel is significantly improved. In addition, the oxygen generation efficiency after ultrasonic and non-ultrasonic treatment was checked by Reynolds dissolved oxygen tester. The results are as follows Fig.11 As shown, A is the amount of oxygen produced under different ultrasonic treatment times, B is the amount of oxygen produced under different room temperature static treatment times, and C is the amount of oxygen produced when treated for 8 minutes under ultrasonic and non-ultrasonic conditions. Fig.11It can be seen that the production of dissolved oxygen is very slow without ultrasonic treatment, and the dissolved oxygen concentration gradually increases after ultrasonic treatment for different time periods.

[0098] Test Example 6

[0099] Characterization of the glucose-lowering and singlet oxygen-generating properties of multifunctional hydrogels

[0100] Take 1 ml of 5 mg / ml glucose in a test tube, add 200 μl of the hydrogel loaded with different concentrations (0, 100, 200, 300, 500 μg / ml) of POM prepared in Example 3, then add 800 μl of pH 6.8 PBS buffer and 100 μl of 3 mmol / l TMB (the solvent is alcohol, TMB is a probe of hydrogen peroxide), and finally add ultrapure water to make the volume 3 ml. 2 ) and reacted for 10 min (2 times, 5 min each time, 3 min interval). After the reaction, the absorbance was measured with a UV-visible spectrophotometer. Each sample was tested 3 times and the average value was taken. The test results of the hypoglycemic effect are shown in Fig.12 As shown, A is the UV-visible absorption spectrum after ultrasonic treatment, B is the UV-visible absorption spectrum without ultrasonic treatment, and C is the maximum absorbance comparison of TMB after ultrasonic treatment and without ultrasonic treatment. Fig.12 It can be seen that the absorption peak of TMB increased greatly after ultrasonic treatment, indicating that a large amount of hydrogen peroxide was produced, indicating a good Gox-like effect.

[0101] Then, the same method was used (other conditions remained unchanged, only the type of probe was changed) to verify the singlet oxygen production efficiency of HP with or without ultrasound using the singlet oxygen green fluorescent probe (SOSG). The results are as follows Fig.13 As shown, A is the fluorescence emission spectrum after ultrasonic treatment, B is the fluorescence emission spectrum without ultrasonic treatment, and C is the ratio change of the maximum fluorescence intensity of SOSG at 521 nm (where F t represents after treatment, and F0 represents before treatment). It can be seen that the ratio gradually increases after ultrasonic treatment, and more singlet oxygen is produced. Fig.13 It can be seen that the efficiency of singlet oxygen generation is greatly improved after ultrasound.

[0102] Test Example 7

[0103] Verification of cytotoxicity of multifunctional hydrogels

[0104] The biosafety of the multifunctional hydrogel was verified by CCK-8. Hydrogels loaded with different concentration gradients (0-1 mg / mL) of POM (prepared in Example 3) were designed, and a PBS group was set as a positive control. RAW264.7 and L929 were inoculated into 96-well plates (10 5 The cells were cultured with complete DMEM medium at 37°C and 5% CO2 for 24 h. Subsequently, hydrogels (hydrogels loaded with different concentrations of POM prepared in Example 3) were added for 4 h. The ultrasound group was given ultrasound and cultured for 24 h. The culture medium was then discarded and 10% CCK-8 solution was added. After 30 min, the absorbance at 450 nm was measured using an enzyme reader to calculate the cell survival rate. The results are shown in FIG. Fig.14 As shown, A is the cell activity of RAW264.7 cells under different conditions, and B is the cell activity of L929 cells under different conditions, indicating that HP is non-toxic to normal cells and immune cells and has excellent biosafety.

[0105] Test Case 8

[0106] Antibacterial properties of multifunctional hydrogels

[0107] Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used as representative bacterial species to evaluate the antibacterial properties of the hydrogel by the plate count method. First, the above bacterial species were cultured in solid Luria-Bertani (LB) medium for 24 hours, and a small amount of colonies were taken with an inoculation loop into 5 ml of broth medium. Then, the culture was shaken and incubated in a constant temperature shaker at 37°C and 200 rpm for 12 hours. When the OD value of the bacterial solution at 450 nm reached 0.5, the 1×10 8 CFU / ml of bacterial suspension was diluted to 1×10 5 CFU / ml for later use. The experiment was divided into four groups: PBS+US, HA+US, HP-US, HP+US ​​(wherein +US represents ultrasonic treatment, -US represents no ultrasonic treatment, HP is the multifunctional hydrogel with a final POM concentration of 0.5 mg / ml prepared in Example 3, and HA is the hydrogel with a POM loading concentration of 0 mg / ml prepared in Example 3). Glucose solution (final concentration of 20 mmol / l) was added to the bacterial solution, and the above bacterial solution was mixed with different hydrogels respectively. The ultrasonic group was subjected to ultrasound (frequency 1.0 MHz, duty cycle 50%, power 1.0 W / cm 2 After 2 times of ultrasound treatment (13 minutes for the group without ultrasound treatment), 10ul of bacterial solution from each group was spread on a solid LB medium plate, placed in a bacterial incubator, and cultured at 37℃ for 12h, and then photographed (such as Fig.15A, unstained upper part in C) and colony counts (as shown in Fig.15 As shown in B, the left picture is S. aureus and the right picture is E. coli).

[0108] The effect of hydrogel on the growth of bacterial biofilm was investigated by crystal violet staining experiment. In a sterile well plate, different hydrogels and bacterial suspensions were added to each well according to the above grouping. The final concentration of glucose was 20mmol / l. After thorough mixing, the well was incubated at 37°C for 48 hours to form a bacterial biofilm. After that, the culture medium was aspirated, the hydrogel was taken out, and washed 3 times with sterile ultrapure water to remove planktonic bacteria and impurities. Add 1% crystal violet solution for staining for 20 minutes. Aspirate the crystal violet solution, continue to wash with sterile ultrapure water, and take pictures of the stained well plate with a camera (such as Fig.15 33 wt% glacial acetic acid was then added to each well for decolorization for 30 min, and the absorbance of each well was measured at 630 nm using an ELISA reader. The absorbance of the PBS+US group was compared with that of the other groups (see Figure 2A). Fig.15 As shown in D in the figure, the ordinate is the absorbance of other groups / the absorbance of PBS+US group×100%, where the left figure is S. aureus and the right figure is E. coli). Crystal violet staining and plate counting method both confirmed that HP has a good bactericidal effect under ultrasound.

[0109] Test Example 9

[0110] Animal experiments on promoting wound recovery

[0111] Eight-week-old male C57BL / 6 mice (17-22g) were selected and adapted to feeding for one week before modeling. First, fast for 12 hours, and intraperitoneally inject STZ (streptozotocin) three times (100mg / kg, 80mg / kg, 60mg / kg), with an interval of 24 hours between each administration, provide 10% sucrose water and ensure sufficient drinking water; one week after the injection, the diabetic mice were successfully modeled if the blood glucose level exceeded 16.1mmol / L twice in 3 days. After that, the back of the mice was shaved and a 10 mm diameter wound was created with a hole punch, and 20μl of Staphylococcus aureus (1×10 7CFU / ml), after the mice were infected with bacteria for 24 hours, the wounds were randomly divided into 5 groups (n=6), and the corresponding materials were added: PBS+US group (100μl, PBS, with ultrasonic treatment), HA+US group (100μl, HA hydrogel with a POM loading concentration of 0mg / ml prepared in Example 3, with ultrasonic treatment), POM+US group (100μl, 0.5mg / ml POM solution, with ultrasonic treatment), HP-US group (100μl, HP hydrogel with a POM final concentration of 0.5mg / ml prepared in Example 3, without ultrasonic treatment), HP+US ​​group (100μl, HP hydrogel with a POM final concentration of 0.5mg / ml prepared in Example 3, with ultrasonic treatment). The day of modeling was day 0, new materials were replaced every two days, and the ultrasonic treatment group was treated with ultrasound on the 3rd and 7th days respectively (ultrasound parameters: frequency 1.0MHz, duty cycle 50%, power 1.0W / cm 2 , time 10 minutes, 5 minutes each time, 3 minutes interval). During the treatment, the weight, wound pictures and blood sugar data of the mice were recorded every two days. Fig.16 As shown, A is a representative digital photo of wound healing in different groups (PBS+US, HA+US, POM+US, HP-US, HP+US) on different days; B is a schematic diagram of wound healing; C is the wound healing rate of each group on the 7th and 14th days; D is the blood sugar change curve on different days; E is the bacterial colony analysis on the wound surface of mice after treatment. Fig.16 It can be seen that after the multifunctional hydrogel treatment, the wound recovery of mice was improved to a certain extent compared with the PBS group, but the wounds of mice treated with ultrasound healed more quickly (e.g. Fig.16 After 14 days of measurement, it can be seen that the wound area of ​​the HP+US ​​group recovered the best (as shown in A and B in FIG). Fig.16 C in the figure), and Fig.16 As can be seen from D, blood sugar levels will decrease after treatment and remain for a period of time, and blood sugar levels will continue to rise slowly after treatment, but they are still lower than those in other groups. Afterwards, bacteria from the wounds of treated mice were cultured and it was found that there was almost no bacterial growth in the HP+US ​​group (e.g. Fig.16 ).

[0112] The embodiments described are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An ultrasound-stimulated multifunctional hydrogel, characterized in that: The invention comprises an injectable hydrogel carrier and reduced molybdate loaded on the injectable hydrogel carrier; the injectable hydrogel carrier is obtained by amination hyaluronic acid and aldehyde hyaluronic acid through Schiff base covalent cross-linking reaction.

2. The ultrasound-stimulated multifunctional hydrogel according to claim 1, characterized in that: The preparation method of the reduced molybdate comprises: mixing a molybdate aqueous solution with a disodium hydrogen phosphate aqueous solution to obtain a mixed solution; adding L-ascorbic acid to the mixed solution to carry out a reduction reaction; and after the reaction is completed, sequentially subjecting the mixed solution to ethanol sedimentation, centrifugation, washing, dialysis and freeze drying to obtain the reduced molybdate.

3. The ultrasound-stimulated multifunctional hydrogel according to claim 2, characterized in that: The molybdate is ammonium heptamolybdate; the molar ratio of molybdate to disodium hydrogen phosphate in the mixed solution is 1:0.6-2.5; the molar ratio of molybdate contained in the mixed solution to the added L-ascorbic acid is 1:0.1-3.

6.

4. The method for preparing the ultrasonically stimulated multifunctional hydrogel according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: dissolving reduced molybdate, aminated hyaluronic acid and aldehyde-modified hyaluronic acid in water respectively to obtain reduced molybdate aqueous solution, aminated hyaluronic acid aqueous solution and aldehyde-modified hyaluronic acid aqueous solution; mixing the reduced molybdate aqueous solution with the aminated hyaluronic acid aqueous solution to obtain an aminated hyaluronic acid aqueous solution containing reduced molybdate; mixing the aminated hyaluronic acid aqueous solution containing reduced molybdate with the aldehyde-modified hyaluronic acid aqueous solution, carrying out Schiff base covalent cross-linking reaction, and obtaining the ultrasound-excited multifunctional hydrogel.

5. The preparation method according to claim 4, characterized in that: The concentration of the reduced molybdate aqueous solution is 0-10 mg / ml and is not 0; the volume ratio of the reduced molybdate aqueous solution to the amino hyaluronic acid aqueous solution is 1:2-7; the volume ratio of the amino hyaluronic acid aqueous solution containing reduced molybdate to the aldehyde hyaluronic acid aqueous solution is 0.8-2.5:

1.

6. The preparation method according to claim 4, characterized in that: The preparation method of the amino hyaluronic acid comprises: adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 1-hydroxybenzotriazole in a molar amount of 2.0-6.5 times the molar number of the hyaluronic acid repeating unit in sequence to a hyaluronic acid aqueous solution, adjusting the pH value of the mixed system to 6.0-7.0, stirring and reacting at room temperature for 10-20 hours, and then dialyzing and freeze-drying in sequence to finally obtain the amino hyaluronic acid.

7. The preparation method according to claim 4, characterized in that: The preparation method of the aldehyde-modified hyaluronic acid comprises: adding sodium periodate in a molar amount of 0.6-1.5 times the molar number of the hyaluronic acid repeating unit to a hyaluronic acid aqueous solution, stirring and dissolving at room temperature, then placing in a dark environment and stirring for reaction for 4-8 hours, and then quenching the reaction with excess ethylene glycol, and after the reaction is completed, dialyzing and freeze-drying in sequence to finally obtain the aldehyde-modified hyaluronic acid.

8. The preparation method according to claim 6 or 7, characterized in that: The mass percentage concentration of the hyaluronic acid aqueous solution is 0.8-2.5%, and the molecular weight of the hyaluronic acid therein is 100,000-800,000.

9. Use of the ultrasound-stimulated multifunctional hydrogel according to claim 1 in preparing a dressing for treating chronic inflammatory wounds.

10. The use according to claim 9, characterized in that The chronic inflammatory wound is a chronic inflammatory wound of a diabetic patient.

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