Hydrogel with pH responsiveness and preparation method and application thereof

Abstract

The invention discloses hydrogel with pH responsiveness as well as a preparation method and application of the hydrogel. The preparation method comprises the following steps: stirring and dispersing the hyaluronic acid, the N, N-diisopropylethylamine, the 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and the N-hydroxysuccinimide into water; gentamicin sulfate is added, and stirring is continued; performing freeze-drying; carrying out hydration to form hydrogel; soaking in a mesoporous silica suspension; and soaking in a bromothymol blue solution. According to the invention, hyaluronic acid, gentamicin sulfate, mesoporous silica and bromothymol blue are combined to prepare the hydrogel with pH responsiveness. The hydrogel can release gentamicin sulfate through hydrolysis to effectively resist bacteria, and the color of the loaded pH sensitive indicator bromothymol blue can also generate characteristic change along with the change of the pH value of the wound microenvironment, so that the visual monitoring of the wound state is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a pH-responsive hydrogel and a preparation method and application thereof. BACKGROUND

[0002] In the field of clinical medicine and wound care, real-time monitoring of the pH value of the wound microenvironment is crucial for assessing the healing process, preventing infection, and developing precise treatment plans. Traditional monitoring methods often rely on frequent sample collection and laboratory analysis, which are cumbersome and cannot achieve continuous dynamic monitoring. With the advancement of materials science and biomedical engineering, developing intelligent hydrogel wound dressings with pH responsiveness has become a key direction to break this bottleneck.

[0003] Hyaluronic acid is a non-sulfated glycosaminoglycan that, as a major component of the extracellular matrix of the skin, is deeply involved in the regulation of inflammation, vascular regeneration, and tissue repair processes. Due to its excellent hydrophilicity and biodegradability, hyaluronic acid has been used to produce different types of wound dressings, such as sponges, membranes, and hydrogels. SUMMARY

[0004] To solve the above technical problems, the application provides a pH-responsive hydrogel and a preparation method and application thereof. The application uses hyaluronic acid, gentamicin sulfate, mesoporous silica, and bromothymol blue to prepare a pH-responsive hydrogel. This hydrogel not only releases gentamicin sulfate through hydrolysis, effectively resisting bacteria and reducing the risk of infection, but also uses bromothymol blue as a pH-sensitive indicator that changes from blue to yellow as the pH value of the wound microenvironment decreases. When bacterial infection occurs, the local environment can trigger a characteristic color change in the indicator, achieving real-time visual monitoring of the wound state. The hydrogel provided by the application can not only reduce the pain caused by frequent replacement of traditional dressings for patients, but also provide intuitive feedback on the healing process for clinical diagnosis and treatment, helping to assess the risk of wound infection and optimize treatment plans in a timely manner.

[0005] To achieve the above-mentioned purposes, the technical solutions adopted by the application are as follows:

[0006] On the one hand, the application provides a preparation method of a pH-responsive hydrogel, comprising the following steps:

[0007] (1) Stir and disperse hyaluronic acid, N,N-diisopropylethylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and N-hydroxysuccinimide in water; add gentamicin sulfate and continue stirring; freeze-dry to obtain a hyaluronic acid-gentamicin sulfate freeze-dried hydrogel;

[0008] (2) hydrating the hyaluronic acid-gentamicin sulfate lyophilized hydrogel prepared in step (1) to form a hydrogel; and then soaking the hydrogel in a mesoporous silica suspension to obtain a hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel;

[0009] (3) soaking the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (2) in a bromothymol blue solution to obtain a hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel.

[0010] As a preferred embodiment, in step (1), the mass ratio of the hyaluronic acid, N,N-diisopropylethylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, water and gentamicin sulfate is 0.2-0.3:0.03-0.12:0.05-0.2:0.06-0.08:30-70:0.5-0.6.

[0011] Preferably, in step (1), the stirring and dispersing time is 5-15 minutes.

[0012] Preferably, in step (1), the continuous stirring time is 12-36 hours.

[0013] Preferably, in step (1), the lyophilization temperature is -50 to -70°C.

[0014] As a preferred embodiment, in step (1), the continuous stirring further comprises a dialysis treatment; the dialysis liquid for the dialysis is deionized water; the molecular weight cut-off of the dialysis is 1000-7000; and the dialysis is performed 6-8 times, with the interval time for changing the dialysis liquid being 6-10 hours.

[0015] As a preferred embodiment, in step (2), the preparation method of the mesoporous silica comprises the following steps:

[0016] The cetyltrimethylammonium bromide is added to a mixed solution of ammonia and ethanol, and stirred and reacted; tetraethyl orthosilicate is added, and the stirring and reaction is continued; and the mesoporous silica is obtained after drying and calcination.

[0017] Preferably, the mixed solution of ammonia, ethanol and water is a mixed solution of 0.8-1 mL of ammonia with a concentration of 25%-28% (mass fraction), 50-70 mL of ethanol and 60-100 mL of water.

[0018] Preferably, the mass ratio of the cetyltrimethylammonium bromide, ethanol and tetraethyl orthosilicate is 0.2-0.8:35-60:0.7-1.

[0019] Preferably, the stirring and reaction time is 0.5-1.5 hours.

[0020] Preferably, the time for continuing stirring the reaction is 5-7 hours.

[0021] Preferably, the drying is vacuum drying at 60-100°C for 10-14 hours.

[0022] Preferably, the calcination is calcination at 500-580°C for 4-8 hours.

[0023] In some specific embodiments, the method further comprises a washing treatment before the drying.

[0024] As a preferred embodiment, in step (2), the hydrous process of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel is performed by hydrating 0.2-0.3 g of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel with 10-30 mL of water.

[0025] Preferably, in step (2), the time for hydrating is 12-36 hours.

[0026] As a preferred embodiment, in step (2), the mass ratio of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel to mesoporous silica is 0.2-0.3:0.024-0.096.

[0027] Preferably, in step (2), the concentration of the mesoporous silica suspension is 3-8 mg / mL.

[0028] Preferably, in step (2), the time for soaking is 24-72 hours.

[0029] In some specific embodiments, in step (2), the method further comprises a washing treatment after the soaking.

[0030] As a preferred embodiment, in step (3), the mass ratio of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel to bromothymol blue is 0.5-1:0.008-0.018.

[0031] Preferably, the concentration of the bromothymol blue solution is 1-1.5 mg / mL.

[0032] Preferably, the time for soaking is 12-24 hours.

[0033] In another aspect, the present application provides the hydrogel obtained by the above method.

[0034] In the technical solution of the present application, the hyaluronic acid and gentamicin sulfate form a hydrogel through amide covalent bond and electrostatic interaction; the mesoporous silica is further combined with the hydrogel through electrostatic interaction and hydrogen bond; and the bromothymol blue is loaded on the mesoporous silica through van der Waals force and hydrogen bond.

[0035] In another aspect, the present invention provides the use of the above-mentioned hydrogel in the preparation of wound dressings.

[0036] The beneficial effects of this invention are: the pH-responsive hydrogel constructed by this invention can achieve dual functions of antibacterial and monitoring. On the one hand, the hydrogel carrier hydrolyzes to release gentamicin sulfate to build an antibacterial barrier. On the other hand, the loaded bromothymol blue indicator produces gradient color changes with the change of wound pH, transforming the wound status into a visual signal, reducing the need for frequent dressing changes, and providing dynamic healing process feedback for clinical diagnosis. Attached Figure Description

[0037] Figure 1 The images show scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the mesoporous silica, hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel prepared in Example 1 of this invention, and the SEM image of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel prepared in Example 4.

[0038] Figure 2 The infrared spectra of mesoporous silica, bromothymol blue, and mesoporous silica-bromothymol blue in Example 1 of the present invention are shown.

[0039] Figure 3 Infrared spectra of hyaluronic acid, gentamicin sulfate, hyaluronic acid-gentamicin sulfate lyophilized hydrogel, and hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel in Example 1 of the present invention.

[0040] Figure 4 This is a graph showing the degradation rate of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel at different pH values ​​in Example 1 of this invention.

[0041] Figure 5 This is a graph showing the gentamicin sulfate release curves of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel at different pH values ​​in Example 1 of the present invention.

[0042] Figure 6 This is a schematic diagram of the color development of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel at different pH values ​​and the corresponding ultraviolet-visible spectrum in Example 1 of the present invention.

[0043] Figure 7 This image shows the antibacterial effect of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel on Escherichia coli and Staphylococcus aureus in Example 1 of this invention. Detailed Implementation

[0044] The following examples merely serve to illustrate the application, but are not intended to limit the scope of the application. Therefore, the general description of the application in the examples is not intended to limit the scope of the claimed application, but merely to represent selected embodiments of the application. Based on the examples of the application, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of the application.

[0045] In the present application, all the equipment and raw materials, etc. can be purchased from the market or commonly used in the industry, unless otherwise specified. The methods in the following examples are all conventional methods in the art, unless otherwise specified.

[0046] Example 1:

[0047] The present embodiment provides a kind of hydrogel with pH response, and the preparation method comprises the following steps:

[0048] (1) 0.25 g of hyaluronic acid is dissolved in 50 mL of deionized water, 0.1 mL of N, N-diisopropyl ethylamine, 0.12 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.07 g of N-hydroxysuccinimide are added, and stirred for 10 minutes; then 0.55 g of gentamicin sulfate is added, and stirring is continued for 24 hours; transfer to a dialysis bag with a molecular weight cut-off of 3500, and dialyze with deionized water; dialysis water is replaced six times, with an interval of 8 hours each time; after dialysis, the contents of the dialysis bag are freeze-dried at -60 DEG C to obtain a white fluffy product, which is a hyaluronic acid-gentamicin sulfate freeze-dried hydrogel;

[0049] (2) 0.5 g of cetyltrimethylammonium bromide is weighed; added to a mixed solution prepared from 0.9 mL of 26% (mass fraction) ammonia water, 60 mL of anhydrous ethanol and 80 mL of deionized water; magnetically stirred at room temperature for 1 hour; then slowly drop 0.9 mL of tetraethyl silicate, continue to stir for 6 hours; centrifuge the obtained sample, collect the solid product, and repeatedly wash with deionized water and anhydrous ethanol several times; vacuum dried at 80 DEG C for 12 hours; the dried sample is placed in a muffle furnace at 540 DEG C for 6 hours to remove cetyltrimethylammonium bromide, and mesoporous silica is obtained;

[0050] (3) 0.25 g of hyaluronic acid-gentamicin sulfate freeze-dried hydrogel prepared in step (1) is hydrated with 20 mL of water overnight to form a hydrogel; immersed in 10 mL of 5 mg / mL mesoporous silica suspension, and stand for 48 hours; washed with deionized water several times to obtain a hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel;

[0051] (4) Weigh 0.75g of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (3) and let it stand overnight in 10mL of bromothymol blue solution with a concentration of 1.25mg / mL; wash with deionized water multiple times to remove impurities and obtain hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel.

[0052] In this embodiment, a mesoporous silica material loaded with bromothymol blue was also prepared. The preparation process was as follows: 50 mg of the mesoporous silica prepared in step (2) was weighed and immersed in 10 mL of a bromothymol blue solution with a concentration of 1.25 mg / mL. The mixture was left overnight to obtain the mesoporous silica material loaded with bromothymol blue (mesoporous silica-bromothymol blue).

[0053] Performance testing:

[0054] (1) The scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the mesoporous silica prepared in Example 1 are shown below. Figure 1 As shown in (A) and (B) in the figure, mesoporous silica exhibits a relatively uniform spherical shape and a distinct mesoporous structure. Figure 1 (C) is a scanning electron microscope image of hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel. It can be clearly seen that the formed hydrogel has a three-dimensional network structure containing large cavities. The rich pore structure indicates that the hydrogel has good water absorption and air permeability, which is beneficial for its use as a wound dressing.

[0055] (2) The infrared spectra of mesoporous silica, bromothymol blue, and mesoporous silica-bromothymol blue in Example 1 are as follows: Figure 2 As shown in the figure. It can be seen from the figure that mesoporous silica at 970 cm⁻¹... -1 The characteristic peak at 802 cm⁻¹ is attributed to the bending vibration of Si-O. -1 and 1063cm -1 The characteristic peak at 3429 cm⁻¹ represents the symmetric and asymmetric stretching vibrations of Si-O-Si; bromothymol blue at 3429 cm⁻¹... -1 The characteristic peak at the position is the stretching vibration of -OH. The characteristic peaks of mesoporous silica and bromothymol blue can be observed in the infrared spectrum of mesoporous silica-bromothymol blue, indicating that bromothymol blue is successfully loaded into mesoporous silica.

[0056] (3) The infrared spectra of hyaluronic acid, gentamicin sulfate, hyaluronic acid-gentamicin sulfate lyophilized hydrogel, and hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel in Example 1 are as follows: Figure 3 As shown in the figure, the infrared spectrum of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel shows that hyaluronic acid can be observed at 1411 cm⁻¹. -1asymmetric stretching vibration peak of C=0, gentamicin sulfate at 1620 cm -1 bending vibration peak of -NH, hyaluronic acid-gentamicin sulfate hydrogel at 1650 cm -1 amide C=0 stretching vibration peak appearing at 970 cm -1 bending vibration peak of Si-O, 802 cm -1 and 1063 cm -1 symmetric and asymmetric stretching vibration peaks of Si-O-Si, bromothymol blue at 3429 cm -1 stretching vibration peak of -OH. The results show the successful preparation of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel.

[0057] (4) Take 0.75 g of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel and place it in 50 mL of phosphate buffer solution with pH values of 5.5, 7.4 and 8.0, respectively; place the solution in a 37°C constant temperature water bath, and every time interval, take out the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel, and use filter paper to absorb the surface moisture and record the weight, and finally calculate the degradation rate according to the mass change of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel. The degradation rate curve at pH 8.0 is shown in Figure 4 It can be seen from the figure that the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel can be completely degraded in a shorter time in an alkaline environment with pH 8.0. This is because the hyaluronic acid-gentamicin sulfate hydrogel is mainly formed by covalent amide bonds and electrostatic interactions. In an alkaline condition, hydroxyl ions act as nucleophiles to attack the carbonyl carbon atoms in the amide bond to form a tetrahedral intermediate, thereby accelerating the breakage of the amide bond and destroying the electrostatic interaction. At the same time, hyaluronic acid is more easily degraded in an alkaline condition. These factors make the hyaluronic acid-gentamicin sulfate hydrogel self-degrade faster in a bacterial infected wound, avoiding the secondary damage caused by the removal of the dressing. The degradation rate of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel is slightly slower in neutral conditions with pH 7.4 and acidic conditions with pH 5.5 than in alkaline conditions with pH 8.0, but it can also be completely degraded eventually, indicating that the hydrogel can self-degrade when used in wound treatment, eliminating the pain caused by removal from the wound.

[0058] (5) Take 0.75 g of hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel, and place it in phosphate buffer solutions with pH values of 5.5, 6.0, 6.5, 7.0, 7.5, and 8.0, respectively, and let it stand for 30 minutes. Record the color change and measure the ultraviolet-visible spectra of the different solutions using an ultraviolet spectrophotometer. The color development of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel at different pH values and the ultraviolet-visible spectra are shown in FIG. 5. As can be seen from the figure, as the alkalinity increases, the hydrogel gradually changes from yellow at pH 5.5 to green at pH 7.0, and then further changes to blue at pH 8.0. In addition, as the pH increases, the position of the absorption peak in the ultraviolet-visible spectrum changes significantly, indicating that the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue has a pH-responsive color development function and can achieve wound monitoring. Figure 6

[0059] (6) Take 0.75 g of hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel, and place it in a dialysis bag with a molecular weight cut-off of 3500. Place the dialysis bag in 50 mL of phosphate buffer solutions with pH values of 5.5, 7.4, and 8.0, respectively, and perform constant temperature magnetic stirring at 37°C in a water bath to conduct in vitro drug release experiments. Every 6 hours during the drug release process, take out 4 mL of the solution to determine the amount of released gentamicin sulfate, and simultaneously add 4 mL of fresh phosphate buffer solution. Mix the 4 mL of the solution taken out with 4 mL of an indantrione solution with a concentration of 5 mg / mL, and perform a color development reaction in a 95°C boiling water bath for 15 minutes. After cooling, use an ultraviolet-visible spectrophotometer to measure the characteristic absorption peak intensity of the derivative product at 564 nm, calculate its concentration, and thus calculate the cumulative percentage of gentamicin sulfate release at different times. The release curve measured in this embodiment is shown in FIG. 6. Figure 5 ​The cumulative release percentage of gentamicin sulfate at pH 8.0 was 73.9% when the release equilibrium was reached. The stability of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel depended on the covalent cross-linking of amide bonds and electrostatic interactions. In an alkaline environment, amide bonds are rapidly hydrolyzed and broken. At the same time, the high concentration of hydroxide ions produces a charge shielding effect, weakening the electrostatic interactions between charged groups, and hyaluronic acid is also more easily degraded under alkaline conditions, which is conducive to the hydrolysis of hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue to release gentamicin sulfate for antibacterial action when the wound is infected with bacteria and presents a weak alkaline environment. In addition, the cumulative release percentage of the hydrogel at neutral conditions of pH 7.4 was 60.5%, and the cumulative release percentage of the hydrogel at acidic conditions of pH 5.5 was 53.2%, and the release rate was slightly slower than that at alkaline conditions of pH 8.0, indicating that the hydrogel has pH responsiveness when used in wound healing, and can release more gentamicin sulfate to effectively resist bacteria when the wound is infected and presents alkaline, providing a basis for its application in wound treatment.

[0060] (7) Dilute the E. coli grown to the logarithmic phase to 1 x 10 8 CFU / mL with sterile water; take 1 mg of hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel and add it to a 5 mL centrifuge tube and heat it to 37°C; then add 1 mL of E. coli bacterial solution and mix well, and incubate at 37°C for 40 minutes; take 100 μL of the bacterial suspension and dilute it to 1 x 10 5 CFU / mL, and spread it on an agar plate, and count the number of colonies after 24 hours to evaluate the in vitro antibacterial effect; the antibacterial effect of the hydrogel on S. aureus was also tested by the same method in this example. The graph of the inhibition effect of the hydrogel on E. coli and S. aureus measured in this example is shown in FIG. 2. Figure 7 The inhibition rate of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel on E. coli and S. aureus was 99.2% and 92.4%, respectively.

[0061] Example 2:

[0062] This example provides a hydrogel with pH responsiveness, and the preparation method comprises the following steps:

[0063] (1) 0.2 g of hyaluronic acid was dissolved in 30 mL of deionized water, 0.05 mL of N, N-diisopropyl ethylamine, 0.05 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.06 g of N-hydroxysuccinimide were added, and stirred for 10 minutes; then 0.5 g of gentamicin sulfate was added, and stirring was continued for 24 hours; it was transferred to a dialysis bag with a molecular weight cut-off of 3500, and dialyzed with deionized water; the dialysis water was replaced six times, with an interval of 8 hours each time; after dialysis, the contents of the dialysis bag were freeze-dried at -60°C to obtain a white fluffy product, which was a hyaluronic acid-gentamicin sulfate freeze-dried hydrogel;

[0064] (2) 0.2 g of cetyltrimethylammonium bromide was weighed; it was added to a mixed solution prepared from 0.8 mL of 26% (mass fraction) ammonia water, 50 mL of anhydrous ethanol and 60 mL of deionized water; it was magnetically stirred at room temperature for 1 hour; then 0.8 mL of tetraethyl silicate was slowly added dropwise, and stirring was continued for 6 hours; the resulting sample was centrifuged, and the solid product was collected and repeatedly washed with deionized water and anhydrous ethanol; it was vacuum dried at 80°C for 12 hours; the dried sample was calcined in a muffle furnace at 540°C for 6 hours to remove the cetyltrimethylammonium bromide, and mesoporous silica was obtained;

[0065] (3) 0.2 g of the hyaluronic acid-gentamicin sulfate freeze-dried hydrogel prepared in step (1) was hydrated with 20 mL of water overnight to form a hydrogel; it was immersed in 8 mL of a mesoporous silica suspension with a concentration of 3 mg / mL, and left to stand for 48 hours; it was washed with deionized water several times to obtain a hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel;

[0066] (4) 0.5 g of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (3) was left to stand in 8 mL of a bromothymol blue solution with a concentration of 1.0 mg / mL overnight; it was washed with deionized water several times to remove impurities, and a hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel was obtained.

[0067] Example 3:

[0068] The present embodiment provides a hydrogel with pH responsiveness, and the preparation method comprises the following steps:

[0069] (1) 0.3 g of hyaluronic acid was dissolved in 70 mL of deionized water, 0.15 mL of N, N-diisopropyl ethylamine, 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 0.08 g of N-hydroxysuccinimide were added, and stirred for 10 minutes; then 0.6 g of gentamicin sulfate was added, and stirring was continued for 24 hours; it was transferred to a dialysis bag with a molecular weight cut-off of 3500, and dialyzed with deionized water; the dialysis water was replaced six times, with an interval of 8 hours each time; after dialysis, the contents of the dialysis bag were freeze-dried at -60°C to obtain a white fluffy product, which was a hyaluronic acid-gentamicin sulfate freeze-dried hydrogel;

[0070] (2) 0.8 g of cetyltrimethylammonium bromide was weighed; it was added to a mixed solution prepared from 1.0 mL of 26% (mass fraction) ammonia water, 70 mL of anhydrous ethanol and 100 mL of deionized water; it was magnetically stirred at room temperature for 1 hour; then 1.0 mL of tetraethyl silicate was slowly added dropwise, and stirring was continued for 6 hours; the resulting sample was centrifuged, and the solid product was collected and repeatedly washed with deionized water and anhydrous ethanol; it was vacuum dried at 80°C for 12 hours; the dried sample was calcined in a muffle furnace at 540°C for 6 hours to remove the cetyltrimethylammonium bromide, and mesoporous silica was obtained;

[0071] (3) 0.3 g of the hyaluronic acid-gentamicin sulfate freeze-dried hydrogel prepared in step (1) was hydrated with 20 mL of water overnight to form a hydrogel; it was immersed in 12 mL of a mesoporous silica suspension with a concentration of 8 mg / mL, and left to stand for 48 hours; it was washed with deionized water several times to obtain a hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel;

[0072] (4) 1 g of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (3) was left to stand in 12 mL of a bromothymol blue solution with a concentration of 1.5 mg / mL overnight; it was washed with deionized water several times to remove impurities, and a hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel was obtained.

[0073] Example 4:

[0074] The present embodiment provides a hydrogel with pH responsiveness, and the preparation method comprises the following steps:

[0075] (1) Dissolve 0.25 g of hyaluronic acid in 50 mL of deionized water, add 0.1 mL of N,N-diisopropylethylamine, 0.2 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 0.08 g of N-hydroxysuccinimide, and stir for 10 minutes; then add 0.55 g of gentamicin sulfate and continue stirring for 24 hours; transfer to a dialysis bag with a molecular weight cutoff of 3500 and dialyze with deionized water; change the dialysate six times, with an interval of 8 hours between each change; after dialysis, freeze-dry the contents of the dialysis bag at -60℃ to obtain a white and fluffy product, which is the hyaluronic acid-gentamicin sulfate freeze-dried hydrogel;

[0076] (2) Weigh 0.5 g of hexadecyltrimethylammonium bromide and add it to a mixed solution prepared with 0.9 mL of 26% (mass fraction) ammonia water, 60 mL of anhydrous ethanol and 80 mL of deionized water; stir magnetically at room temperature for 1 hour; then slowly add 0.9 mL of tetraethyl silicate and continue stirring for 6 hours; centrifuge the obtained sample, collect the solid product, and wash it repeatedly with deionized water and anhydrous ethanol several times; vacuum dry at 80 °C for 12 hours; calcine the dried sample in a muffle furnace at 540 °C for 6 hours to remove hexadecyltrimethylammonium bromide and obtain mesoporous silica;

[0077] (3) Weigh 0.25g of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel prepared in step (1) and hydrate it with 20mL of water overnight to form a hydrogel; immerse it in 10mL of mesoporous silica suspension with a concentration of 5mg / mL and let it stand for 48 hours; wash it with deionized water several times to obtain hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel.

[0078] (4) Weigh 0.75g of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (3) and let it stand overnight in 10mL of bromothymol blue solution with a concentration of 1.25mg / mL; wash with deionized water multiple times to remove impurities and obtain hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel.

[0079] The scanning electron microscope (SEM) image of the hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue prepared in this embodiment is shown below. Figure 1 As shown in (D), it can be seen that the morphology of the product in this embodiment has changed compared with that in Example 1. When more coupling agents 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are added, the internal pores of the product hydrogel are reduced, forming a denser network structure.

[0080] The above merely describes the preferred embodiments of the present application, and it should be pointed out that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method for preparing a hydrogel having pH responsiveness, characterized by, The method comprises the following steps: (1) stirring and dispersing hyaluronic acid, N,N-diisopropylethylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide in water; adding gentamicin sulfate and continuing stirring; and obtaining hyaluronic acid-gentamicin sulfate lyophilized hydrogel after lyophilization; (2) hydrating the hyaluronic acid-gentamicin sulfate lyophilized hydrogel prepared in step (1) to form a hydrogel; and obtaining hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel after soaking in a mesoporous silica suspension; (3) obtaining hyaluronic acid-gentamicin sulfate-mesoporous silica-bromothymol blue hydrogel after soaking the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel prepared in step (2) in a bromothymol blue solution.

2. The production method according to claim 1, characterized by, In step (1), the mass ratio of the hyaluronic acid, N,N-diisopropylethylamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, water and gentamicin sulfate is 0.2-0.3:0.03-0.12:0.05-0.2:0.06-0.08:30-70:0.5-0.

6. Preferably, in step (1), the stirring and dispersing is performed for 5-15 minutes. Preferably, in step (1), the continuing stirring is performed for 12-36 hours. Preferably, in step (1), the lyophilization is performed at a temperature of -50 to -70°C.

3. The preparation method according to claim 1, characterized in that, In step (1), the continuing stirring further comprises a dialysis treatment; the dialysis liquid is deionized water; the molecular weight cut-off of the dialysis is 1000-7000; and the dialysis is performed for 6-8 times at an interval of 6-10 hours.

4. The production method according to claim 1, characterized by, In step (2), the method for preparing the mesoporous silica comprises the following steps: adding cetyltrimethylammonium bromide to a mixed solution of ammonia and ethanol, stirring and reacting; adding tetraethyl orthosilicate, continuing stirring and reacting; and obtaining the mesoporous silica after drying and calcining.

5. The preparation method according to claim 4, characterized in that, The mixed solution of ammonia and ethanol is a mixed solution of 0.8-1 mL of ammonia with a concentration of 25%-28%, 50-70 mL of ethanol and 60-100 mL of water; Preferably, the mass ratio of the cetyltrimethylammonium bromide, ethanol and tetraethyl orthosilicate is 0.2-0.8:35-60:0.7-1; Preferably, the stirring and reacting is performed for 0.5-1.5 hours; Preferably, the continuing stirring and reacting is performed for 5-7 hours; Preferably, the drying is performed at 60-100°C for 10-14 hours under vacuum; Preferably, the calcining is performed at 500-580°C for 4-8 hours.

6. The method of claim 1, wherein, In step (2), 0.2-0.3 g of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel is hydrated with 10-30 mL of water. Preferably, in step (2), the hydrating is performed for 12-36 hours.

7. The preparation method according to claim 1, characterized in that, In step (2), the mass ratio of the hyaluronic acid-gentamicin sulfate lyophilized hydrogel to mesoporous silica is 0.2-0.3:0.024-0.096; Preferably, the concentration of the mesoporous silica suspension in step (2) is 3-8 mg / mL; Preferably, the soaking time in step (2) is 24-72 hours.

8. The method of claim 1, wherein, In step (3), the mass ratio of the hyaluronic acid-gentamicin sulfate-mesoporous silica hydrogel to bromothymol blue is 0.5-1:0.008-0.018; Preferably, the concentration of the bromothymol blue solution in step (3) is 1-1.5 mg / mL; Preferably, the soaking time in step (3) is 12-24 hours.

9. The hydrogel obtained by the preparation method of any one of claims 1-8.

10. Use of the hydrogel of claim 9 in the preparation of a wound dressing.

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