A hydrogel releasing acid in stages and long-acting alkali consumption and a preparation method and application thereof

Abstract

The application belongs to the technical field of medical materials, and discloses a hydrogel for graded release of acid and long-acting consumption of alkali as well as a preparation method and application thereof, which comprises the following steps: adding a quaternary ammonium salt chitosan solution into a polyvinyl alcohol-organic acid ester solution, stirring uniformly to obtain a polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution; under stirring, adding a nano metal sulfide colloidal solution into the polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution, stirring uniformly, and repeatedly freezing and thawing to obtain the hydrogel for graded release of acid and long-acting consumption of alkali after thawing. The application proposes a strategy for graded release of acid and long-acting slow release of alkali, the hydrogel slowly releases acid in application, and the process of long-acting consumption of alkali liquid avoids secondary damage of an acid-alkali reaction to a cornea, so that the hydrogel has a good application prospect in clinical application of corneal alkali burn and opens up a new way for application of H2S in corneal alkali burn.

Description

Technical Field

[0001] This invention belongs to the field of medical materials technology, specifically relating to a hydrogel that releases acid in stages and consumes alkali over a long period of time, as well as its preparation method and application. Background Technology

[0002] Alkali burns of the cornea are one of the most common conditions seen in ocular emergencies. It has been reported that 70% of severe chemical burns to the eye are caused by alkali burns, and their treatment is among the most challenging. Alkali burns are primarily caused by alkaline substances such as sodium hydroxide, quicklime, and ammonia. These alkaline agents are lipophilic, capable of dissolving fat and protein in the eye. Upon contact with tissues, they rapidly flow into the eye, continuously damaging corneal epithelial cells, leading to cell decomposition and necrosis, causing corneal opacity, ulceration, or perforation, and in severe cases, even resulting in vision loss. Clinical treatment of corneal alkali burns typically begins with copious irrigation with sterile saline or glucose solution to remove alkali from the ocular surface. Treatment options, such as medication, surgical debridement, or corneal transplantation, are then selected based on the severity of the damage. While these strategies play a crucial role in treating corneal alkali burns, they still present limitations and risks, including invasiveness, limited efficacy, potential ocular complications from drug side effects, and immune rejection. For example, the use of copious solutions in pretreatment can cause ocular discomfort while failing to completely remove alkali; adjusting pH using saline and weak acid solutions in a short time can cause a violent neutralization reaction, resulting in secondary corneal damage; treatment with steroids and methotrexate involves complex application procedures and can lead to complications; surgical debridement carries the risk of secondary corneal damage; and corneal transplantation presents risks such as immune rejection. In recent years, researchers have focused on using biocompatible and biosafety-friendly hydrogels as carriers or scaffolds to load drugs, exosomes, or stem cells to promote corneal epithelial repair. For example, patent CN116606457A utilizes sodium alginate and divalent cations to crosslink and form a hemispherical gel for treating corneal alkali burns. Patent CN116327742A uses hyaluronic acid and other polymers to load dexamethasone and antimicrobial peptides to prepare a multifunctional nanomembrane for treating corneal alkali burns and infections. Patent CN110522947B uses chitosan thermosensitive hydrogel to encapsulate limbal stem cells for treating corneal damage. While these methods have some efficacy in treating corneal alkali burns, the later treatment results are unsatisfactory, with drawbacks such as scar formation and long treatment cycles. Furthermore, they suffer from complex manufacturing methods, low drug loading rates, low cell and derivative survival rates, high costs, and slow drug efficacy. More importantly, these common treatment strategies mainly target the repair of lesions after corneal alkali burns, while ignoring the alkali residue still remaining in the cornea, thus failing to fundamentally inhibit the damage caused by alkali to the eye tissues.Studies have shown that even when the eyes are rinsed with large amounts of saline for 10-120 minutes, the pH of the corneal microenvironment remains at 8.0-12.0. The residual alkali reduces the therapeutic effect and continues to erode the deep tissues of the eye, causing more serious damage to the cornea and other eye tissues (LAUX U, ROTH HW, KREY H, et al. Aqueous humor pH in experimental lye burns and influence of different treatment measures (author's transl) [J]. Albrecht von Graefes Archiv furklinische und experimentalelle Ophthalmologie Albrecht von Graefe's archive for clinical and experimental ophthalmology, 1975, 195(1):33-40).

[0003] Weakly acidic hydrogen sulfide (H2S) is widely considered a signaling gaseous neurotransmitter with excellent antioxidant and anti-inflammatory effects. It has been shown to have significant effects on the retina, cataracts, and glaucoma, reducing oxidative stress damage, regulating apoptosis, and decreasing inflammation. Therefore, applying H2S to the treatment of alkali-induced corneal burns can effectively neutralize alkali that has penetrated deep into the ocular tissues and inhibit the expression of inflammatory factors. However, as a gaseous signaling molecule, H2S is colorless and volatile, limiting its direct clinical application. Currently, exogenous H2S donors, such as water-soluble sulfides NaHS and Na2S, are widely used in basic research, but these donors have limitations and shortcomings, such as unstable release, excessive release, or uncontrolled release time. Therefore, there is an urgent need for a safe, non-toxic hydrogel that can gently and effectively consume residual alkali for rapid treatment of alkali-induced corneal burns, which shows great promise. Summary of the Invention

[0004] In order to overcome the shortcomings and disadvantages of existing treatment methods, the primary objective of this invention is to provide a method for preparing a hydrogel that releases acid in stages and consumes alkali over a long period of time.

[0005] Another object of the present invention is to provide a hydrogel that releases acid in stages and consumes alkali in a long-term manner, prepared by the above method.

[0006] Another object of the present invention is to provide the application of the above-mentioned hydrogel that releases acid in stages and consumes alkali over a long period of time.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for preparing a hydrogel that releases acid in stages and consumes alkali over a long period of time includes the following steps:

[0009] (1) Add quaternary ammonium salt chitosan solution to polyvinyl alcohol-organic acid acid ester solution and stir evenly to obtain polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution.

[0010] (2) Under stirring conditions, the nano metal sulfide colloidal solution was added to the polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution, stirred evenly, and repeatedly frozen and thawed. After thawing, a hydrogel that releases acid in stages and consumes alkali for a long time was obtained.

[0011] Preferably, the polyvinyl alcohol-organic acid ester compound in step (1) is prepared by the following method:

[0012] A certain mass of polyvinyl alcohol is dissolved in water to obtain a polyvinyl alcohol solution. An organic acid solution is slowly added dropwise, and after stirring evenly, a tin tetrachloride catalyst solution is added. The mixture is heated in an oil bath at 40–90°C for 12–72 hours. After sedimentation, dialyzing, and drying, a polyvinyl alcohol-organic acid ester is obtained.

[0013] Preferably, the mass-to-volume ratio of polyvinyl alcohol to water (g / mL) is 1–3:10–50;

[0014] The organic acid is at least one of malic acid, maleic acid, citric acid and fumaric acid, and the organic acid solution is prepared by mixing organic acid and water at a mass-volume ratio of 0.5-4:5-20 g / mL.

[0015] Preferably, the volume ratio of the added organic acid solution to the polyvinyl alcohol solution is 1-5:1-10.

[0016] Preferably, the organic acid solution is stirred until homogeneous for 1 to 2 hours.

[0017] Preferably, the tin tetrachloride solution is prepared by mixing tin tetrachloride and water at a mass-to-volume ratio of 0.01–0.1:5–20 g / mL.

[0018] Preferably, the volume ratio of tin tetrachloride solution to polyvinyl alcohol / organic acid solution is 0.1-1:5-15.

[0019] Preferably, the sedimentation is carried out by sedimentation with an ethanol solution for 12 to 48 hours, wherein the volume fraction of the ethanol solution is 50% to 90%.

[0020] Preferably, the dialysis bag used for dialysis has a molecular weight cutoff of 500-3000D, and the dialysis time is 1-3 days.

[0021] Preferably, the drying temperature is 40–80°C and the drying time is 8–24 hours.

[0022] Preferably, in step (1), the mass-to-volume ratio of polyvinyl alcohol-organic acid ester solution to water is 0.5-2.5:10-30 (g / mL).

[0023] The mass-to-volume ratio of quaternary ammonium chitosan to water in the quaternary ammonium chitosan solution is 0.1–2.0:10–30 (g / mL).

[0024] Preferably, the quaternary ammonium salt chitosan has at least one degree of substitution selected from 92%, 95%, and 98%; the average molecular weight of the quaternary ammonium salt chitosan is 3000-5000.

[0025] Preferably, in step (1), the polyvinyl alcohol-organic acid ester solution requires stirring for 1 to 3 hours to dissolve the polyvinyl alcohol-organic acid ester in water, and the quaternary ammonium salt chitosan solution requires stirring for 0.5 to 2 hours to dissolve the quaternary ammonium salt chitosan in water;

[0026] The volume ratio of the polyvinyl alcohol-organic acid ester solution to the quaternary ammonium salt chitosan solution is 5-15:1-5, and the two are stirred for 10-30 minutes after mixing.

[0027] Preferably, the volume ratio of the nano-metal sulfide colloidal solution and the polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution in step (2) is 0.1-1:10-30.

[0028] Preferably, the colloidal solution of nano-metal sulfide in step (2) is obtained by dispersing nano-metal sulfide particles in an ethanol-water mixture, wherein the mass-volume ratio of nano-metal sulfide, ethanol and water is (0.001-0.01) g: (5-10) mL: (5-10) mL;

[0029] The nano-metal sulfide is at least one of zinc sulfide, iron tetrasulfide, ferrous sulfide, and manganese sulfide.

[0030] Preferably, the freezing temperature for repeated freeze-thaw cycles in step (2) is -20 to -80°C, the freezing time is 4 to 24 hours, and the number of freeze-thaw cycles is 2 to 5.

[0031] The thawing temperature is 25–37°C, and the thawing time is 2–12 hours.

[0032] Preferably, the time for mixing evenly in step (2) is 0.5 to 5 hours.

[0033] Preferably, the polyvinyl alcohol is at least one of type 1788, type 1792 and type 2488, and the average molecular weight of the polyvinyl alcohol is 84,000 to 124,000 and the fineness is 80 to 100 mesh.

[0034] A hydrogel that releases acid in stages and consumes alkali over a long period of time is prepared by the method described above.

[0035] The above-mentioned hydrogel is used in the preparation of materials for alkali burn corneal wounds.

[0036] This invention utilizes green, safe, and abundant small-molecule organic acids to graft and modify polyvinyl alcohol polymers. Polyvinyl alcohol-organic acid esters and biocompatible, inexpensive chitosan derivatives are used as the hydrogel framework, with nano-metal sulfides serving as crosslinking agents and fillers. This process prepares a hydrogel capable of graded release of acid and long-term alkali consumption. The hydrogel prepared by this method can gradedly release small-molecule organic acids and H2S to slowly neutralize alkali solutions that have penetrated to the ocular surface and deep ocular tissues. Simultaneously, this hydrogel possesses good anti-inflammatory and antioxidant properties, good antibacterial properties, and excellent biocompatibility. This invention can be applied to the cornea of ​​patients with alkali burns, providing graded and sustained release of organic acids and H2S, gently and long-term consuming alkali solutions that have penetrated to the ocular surface and deep ocular tissues. It inhibits the continuous damage of alkali to the cornea from the root cause and exerts good anti-inflammatory and antioxidant effects, enhancing the repair of corneal lesions and effectively promoting the healing of alkali-burned corneas.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] (1) In this invention, metal sulfides are used as H2S donors. Small molecule organic acids are grafted onto polymer chains by esterification reaction. In the microenvironment of alkali-burned cornea, the small molecule organic acids generated by the slow hydrolysis and ionization of esters directly remove residual alkali from the cornea. The other part of the organic acids reacts with metal sulfides to generate penetrating and soluble H2S and divalent metal ions. The former is used to consume the alkali that has penetrated into deep tissues, gently regulate the pH of the corneal microenvironment, and inhibit further damage to the cornea and other tissues from the root. The latter divalent metal ions effectively inhibit the matrix metalloproteinases generated in the cornea after alkali burn and play an anti-inflammatory and antioxidant role, which is conducive to the rapid repair of alkali-burned cornea.

[0039] (2) The present invention proposes a strategy of graded acid release and long-term sustained alkali release to make up for the shortcomings of clinical practice which mainly focuses on the repair of alkali burn lesions and ignores the continuous damage to the cornea caused by residual alkali. Furthermore, the hydrogel slowly releases acid in grades during application and the long-term consumption of alkali avoids the secondary damage to the cornea caused by violent acid-base reactions.

[0040] (3) The hydrogel of the present invention has excellent therapeutic effect on alkali-burned cornea. Attached Figure Description

[0041] Figure 1 The image shows the infrared spectrum of PVA-CA in Example 1.

[0042] Figure 2 (ab) shows the actual images of the hydrogels prepared in Example 1 and Comparative Example 1 (Fig. a) and the compressive stress-strain diagrams of the two hydrogels (Fig. b).

[0043] Figure 3 The antibacterial properties of each hydrogel are shown in Figures a and b, which are bar charts of the antibacterial zones and corresponding antibacterial ring widths of the hydrogels prepared in Examples 1, 1 and 2, respectively; Figures c and d are bar charts of the antibacterial effects and corresponding antibacterial rates of the hydrogels prepared in Examples 4, 5 and 6.

[0044] Figure 4 The images show scanning electron microscope (SEM) images of the PMQ-Fe3S4 hydrogels prepared in Examples 4, 5, and 6. The contents of iron tetrasulfide in PMQ-Fe3S4-a, PMQ-Fe3S4-b, and PMQ-Fe3S4-c are 0.33 mg, 0.66 mg, and 0.99 mg, respectively.

[0045] Figure 5 The malic acid sustained-release curve (Fig. a), the cumulative release curve of zinc sulfide (Fig. b), and the pH change curve of the buffer in which the gel is located are shown in Fig. c for the hydrogels prepared for Examples 4, 5, and 6 that stagedly release acid and long-lasting alkali consumption.

[0046] Figure 6 The in vitro citric acid sustained-release curve (Fig. a), the cumulative release curve of zinc sulfide (Fig. b), and the pH change curve of the buffer in which the gel is located are shown in Fig. c for the hydrogels prepared for Examples 7, 8, and 9, which are staged acid release and long-lasting alkali consumption, respectively. The zinc sulfide contents in PCQ-ZnS-a, PCQ-ZnS-b, and PCQ-ZnS-c are 0.42 mg, 0.56 mg, and 0.70 mg, respectively.

[0047] Figure 7 The image shows the effect of different hydrogels applied to a mouse alkali burn model, as shown in Example 6. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. For process parameters not specifically noted, conventional techniques can be referred to.

[0049] Several healthy female C57BL / 6 mice, each weighing approximately 25g, were purchased from the Guangdong Provincial Laboratory Animal Center (Guangzhou, China).

[0050] Example 1

[0051] (1) Weigh 1g of polyvinyl alcohol (PVA) powder of type 1788 with an average molecular weight of 84,000 and passing through an 80-mesh sieve at room temperature, dissolve it in 10mL of water, and stir for 1h to obtain a PVA solution.

[0052] (2) Weigh 1g of citric acid (CA) and dissolve it in 10mL of water to obtain a citric acid solution. Take 5mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 0.5mL / min. Stir for 1h.

[0053] (3) Weigh 0.01g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 0.3mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 40℃ for 12h.

[0054] (4) Add the product from (3) to 50% ethanol and let it settle for 12 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 500D and dialyze for 1 day.

[0055] (5) The product in (4) was transferred to an oven at 40°C and dried for 8 hours to obtain the grafted modified polyvinyl alcohol-citric acid ester (PVA-CA).

[0056] (6) Dissolve 0.5g of the product from (5) in 10mL of water and stir for 1h to obtain a PVA-CA solution.

[0057] (7) Weigh 0.1g of quaternary ammonium salt chitosan (QCS) and dissolve it in 10mL of water. Stir for 0.5h to obtain quaternary ammonium salt chitosan solution. Take 2mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 10min to obtain PVA-CA / QCS solution.

[0058] (8) 0.001 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 5 min to obtain zinc sulfide colloidal solution.

[0059] (9) While stirring, 0.12 mL of zinc sulfide colloidal solution (8) was slowly added dropwise to the solution (7) and stirred for 0.5 h. Then, the solution was transferred to -20 °C for 4 h and thawed at 25 °C for 2 h. The freeze-thaw cycle was repeated twice to obtain a hydrogel (PCQ-ZnS) that loads zinc sulfide nanoparticles, releases acid in stages, and consumes alkali in a long-term alkali-consuming manner.

[0060] A small amount of PVA-CA from step (5) and raw materials CA and PVA were ground into powder, compressed into tablets with potassium bromide, and then subjected to infrared detection to obtain the infrared spectra of PVA-CA and raw materials CA and PVA. Figure 1 CA at 1760cm -1 and 1710cm -1 The characteristic peak at 3300-3500 cm⁻¹ is attributed to the C=O groups in the two carboxyl groups in the structure. -1 The two peaks within the range are attributed to the characteristic -OH peaks in the structure; PVA at 1741 cm⁻¹ -1The characteristic peak at 1710 cm⁻¹ is attributed to un-hydrolyzed ester bonds present in PVA. This is similar to the peak observed in PVA at 1710 cm⁻¹. -1 Compared to the characteristic peak at 1739 cm⁻¹, PVA-CA has a peak at 1739 cm⁻¹. -1 The proportion of carbonyl peaks in the infrared spectrum increases, and the increase is due to the characteristic carbonyl peaks on the ester bonds formed after grafting PVA and CA; in addition, the C=O peak of PVA-CA shifts to 1633 cm⁻¹. -1 This is because the -COOH in CA undergoes an esterification reaction with the -OH in PVA, resulting in a C=O shift. The above analysis indicates that citric acid has successfully bonded to polyvinyl alcohol.

[0061] Example 2

[0062] Unlike Example 1, in step (8), 0.005 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 5 min to obtain zinc sulfide colloidal solution, and finally a hydrogel (PCQ-ZnS) loaded with nano-metal sulfide was obtained by graded release of acid and long-term consumption of alkali.

[0063] Example 3

[0064] Unlike Example 1, in step (8), 0.01 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 5 min to obtain zinc sulfide colloidal solution, and finally a hydrogel (PCQ-ZnS) loaded with nano-metal sulfide was obtained by graded release of acid and long-term consumption of alkali.

[0065] Comparative Example 1

[0066] (1) Weigh 1g of polyvinyl alcohol (PVA) powder of type 1788 with an average molecular weight of 84,000 and passing through an 80-mesh sieve at room temperature, dissolve it in 10mL of water, and stir for 1h to obtain a PVA solution.

[0067] (2) Weigh 1g of citric acid (CA) and dissolve it in 10mL of water to obtain a citric acid solution. Take 5mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 0.5mL / min. Stir for 1h.

[0068] (3) Weigh 0.01g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 0.3mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 40℃ for 12h.

[0069] (4) Add the product from (3) to 50% ethanol and let it settle for 12 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 500D and dialyze for 1 day.

[0070] (5) The product in (4) was transferred to an oven at 40°C and dried for 8 hours to obtain the grafted modified polyvinyl alcohol-citric acid ester (PVA-CA).

[0071] (6) Dissolve 0.5g of the product from (5) in 10mL of water and stir for 1h to obtain a PVA-CA solution.

[0072] (7) Weigh 0.1g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 0.5h to obtain quaternary ammonium salt chitosan solution. Take 2mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 10min to obtain PVA-CA / QCS solution.

[0073] (8) The product from (7) was transferred to -20℃ for 4 hours and thawed at 25℃ for 2 hours. The product was frozen and thawed twice to obtain the acid-releasing hydrogel PVA-CA / QCS (PCQ).

[0074] Comparative Example 2

[0075] (1) Weigh 1g of polyvinyl alcohol (PVA) powder of type 1788 with an average molecular weight of 84,000 and passing through an 80-mesh sieve at room temperature, dissolve it in 10mL of water, and stir for 1h to obtain a PVA solution.

[0076] (2) Add the product from (1) to 50% ethanol and let it settle for 12 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 500D and dialyze for 1 day.

[0077] (3) The product from (2) was transferred to a 40°C oven and dried for 8 hours to obtain purified polyvinyl alcohol (PVA).

[0078] (4) Dissolve 0.5g of the product from (3) in 10mL of water and stir for 1h to obtain a PVA solution.

[0079] (5) Weigh 0.1g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 0.5h to obtain quaternary ammonium salt chitosan solution. Take 2mL of quaternary ammonium salt chitosan solution and add it to the solution in (6) and stir for 10min.

[0080] (6) The product from (5) was transferred to -20℃ for 4 hours and thawed at 25℃ for 2 hours. The product was frozen and thawed twice to obtain PVA-QCS(PQ) hydrogel.

[0081] Test Example 1

[0082] The actual samples of the PCQ hydrogel of Comparative Example 1 and the PCQ-ZnS hydrogel of Example 1 are shown below. Figure 2 As shown in (a), both gels are opaque. The columnar PCQ gel structure collapses somewhat, while the gel with added zinc sulfide nanoparticles is more robust. Mechanical properties of the two gels were tested. Figure 2In (b), zinc sulfide nanoparticles were added in Example 1, and the compressive strength of the gel was significantly greater than that of the gel in Comparative Example 1 without zinc sulfide, thus proving that the addition of zinc sulfide nanoparticles can improve the mechanical properties of the gel.

[0083] Test Example 2

[0084] (1) Prepare liquid culture medium by dissolving 2g of nutrient broth in 100mL of ultrapure water, sterilizing it in an autoclave, and cooling it to room temperature for later use.

[0085] (2) Inoculate Escherichia coli (TACC35218) into (1) and incubate at 37℃ and 120 rpm for a certain period of time. Measure the optical density of the bacterial solution periodically using an ELISA reader. Take a bacterial solution with a concentration of 10... 4 CFU / mL available for use.

[0086] (3) Prepare solid culture medium by dissolving 20g of nutrient broth and 32g of agar powder in 1000mL of ultrapure water, sterilizing in an autoclave, and cooling the temperature to 60℃ for later use.

[0087] (4) Take out circular hydrogels with a diameter of 10 mm from the hydrogel loaded with zinc sulfide (Example 1 PCQ-ZnS), the hydrogel without nano-sulfide loading (Comparative Example 1 PCQ), and the hydrogel constructed only from polyvinyl alcohol and quaternary ammonium salt chitosan (Comparative Example 2 PQ) for later use.

[0088] (5) Pour the solid culture of (3) into multiple culture dishes, each containing about 15 mL of culture medium. After cooling and solidification, inoculate 200 uL of Escherichia coli (TACC35218) in (2) into the solid culture medium and apply bacterial agent.

[0089] (6) Add the circular hydrogel obtained in (4) to the solid culture medium in (5) and incubate in a constant temperature incubator at 37℃ for 24h. Observe the inhibition zone around the gel, measure the diameter of the inhibition zone and the diameter of the hydrogel with a vernier caliper, and calculate the width of the inhibition zone according to formula (1).

[0090] N=(Dd) / 2(1)

[0091] Where N represents the width of the inhibition zone (mm); D represents the diameter of the inhibition zone (mm); and d represents the diameter of the hydrogel on the culture medium (mm), where d = 10 mm.

[0092] Using the same method, Staphylococcus aureus (ATCC25923) was inoculated in step (2), and the antibacterial activity of the hydrogel against Staphylococcus aureus was tested.

[0093] Test results as follows Figure 3As shown in (a) and (b), the graded release acid hydrogel loaded with zinc sulfide has the largest inhibition zone width against Escherichia coli and Staphylococcus aureus, and has obvious antibacterial properties.

[0094] Example 4

[0095] (1) Weigh 3g of polyvinyl alcohol (PVA) powder of type 2488 with an average molecular weight of 120,000 and passing through a 100-mesh sieve at room temperature, dissolve it in 50mL of water, and stir for 1h to obtain a PVA solution.

[0096] (2) Weigh 4g of malic acid (MA) and dissolve it in 20mL of water to obtain a malic acid solution. Take 10mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 2mL / min. Stir for 2h.

[0097] (3) Weigh 0.1g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 6mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 70℃ for 24h.

[0098] (4) Add the product from (3) to 70% ethanol and let it settle for 48 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 3000D and dialyze for 3 days.

[0099] (5) The product in (4) was transferred to a 60°C oven and dried for 8 hours to obtain grafted modified polyvinyl alcohol-malate (PVA-MA).

[0100] (6) Dissolve 2g of the product from (5) in 30mL of water and stir for 3h to obtain a PVA-MA solution.

[0101] (7) Weigh 2g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 2h to obtain quaternary ammonium salt chitosan solution. Take 3mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 30min to obtain PVA-MA / QCS solution.

[0102] (8) 0.002 g of iron tetrasulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 10 min to obtain iron tetrasulfide colloidal solution.

[0103] (9) While stirring, 1.65 mL of the triferrote tetrasulfide colloidal solution of (8) was slowly added dropwise to the solution of (7) and stirred for 4 h. Then, it was transferred to -80℃ for freezing for 24 h and thawed at 37℃ for 12 h. A total of 5 freeze-thaw cycles were performed to obtain a hydrogel PVA-MA / QCS-Fe3S4 (PMQ-Fe3S4-a) loaded with 0.33 mg triferrote tetrasulfide, which releases acid in stages and consumes alkali in a long-term alkali-consuming manner.

[0104] Example 5

[0105] Unlike Example 4, in step (8), 0.004 g of iron tetrasulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 10 min to obtain an iron tetrasulfide colloidal solution, and finally a hydrogel PVA-MA / QCS-Fe3S4 (PMQ-Fe3S4-b) loaded with 0.66 mg of iron tetrasulfide, which is a stepwise acid-releasing, long-lasting alkali-consuming hydrogel.

[0106] Example 6

[0107] Unlike Example 4, in step (8), 0.006 g of iron tetrasulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 10 min to obtain an iron tetrasulfide colloidal solution, and finally a hydrogel PVA-MA / QCS-Fe3S4 (PMQ-Fe3S4-c) loaded with 0.99 mg of iron tetrasulfide, which is a stepwise acid-releasing, long-lasting alkali-consuming hydrogel.

[0108] Test Example 3

[0109] (1) Prepare liquid culture medium by dissolving 2g of nutrient broth in 100mL of ultrapure water, sterilizing it in an autoclave, and cooling it to room temperature for later use.

[0110] (2) Inoculate Escherichia coli (TACC35218) into (1) and incubate at 37℃ and 120 rpm for a certain period of time. Measure the optical density of the bacterial solution periodically using an ELISA reader. Take a bacterial solution with a concentration of 10... 4 CFU / mL available for use.

[0111] (3) Prepare solid culture medium. Take 20g of nutrient broth and 32g of agar powder and dissolve them in 1000mL of ultrapure water. Put them in an autoclave for sterilization. When the temperature drops to 60℃, pour them into multiple petri dishes. Each petri dish contains about 15mL of culture medium. Let it cool and solidify before use.

[0112] (4) Take out 1 mg of hydrogel from each of the hydrogels loaded with 0.33 mg, 0.66 mg and 0.99 mg of iron tetrasulfide (Examples 4-6: PMQ-Fe3S4-a, PMQ-Fe3S4-b and PMQ-Fe3S4-c) for later use.

[0113] (5) Dilute the bacterial solution in (2) by 100 times, and then take 100 μL into each well of a 96-well plate. Add the hydrogel obtained in (4) into the 96-well plate containing the bacterial solution, and incubate in a constant temperature incubator at 37°C for 24 hours.

[0114] (6) Dilute the bacterial culture co-cultured with each hydrogel in the 96-well plate of (5) by 1000 times, take 50 μL and inoculate it into the solid culture medium of (3), spread the bacterial agent, and incubate it in a constant temperature incubator at 37℃ for 24 h. In the same way, Staphylococcus aureus (ATCC25923) was inoculated in step (2), and the antibacterial activity of the hydrogel against Staphylococcus aureus was tested.

[0115] (7) Compare the colony counts in the solid culture medium, such as Figure 3 As shown in (cd), the graded release acid hydrogel loaded with iron tetrasulfide has a significant antibacterial effect on Escherichia coli, and the antibacterial rate of the hydrogel increases with the increase of iron tetrasulfide content.

[0116] Test Example 4

[0117] (1) The gels from Examples 4, 5, and 6 were placed in dialysis bags (MWCO = 500D) and placed in phosphate buffer solution at pH = 9.0. Three parallel groups were set up and incubated at 37°C. 2 mL of sample was taken out at intervals (0h, 1h, 3h, 5h, 8h, 12h, 18h, 24h, 36h, 48h, 60h, 72h), and an equal volume of fresh buffer solution was added. 1 g of 2,7-naphthol was dissolved in 100 mL of sulfuric acid. 1 mL of sample from (1) was taken and 6 mL of 2,7-naphthol solution was added. The mixture was heated in a water bath at 100°C for 20 min. After cooling to near room temperature, the absorbance was measured colorimetrically under 385 nm UV light. The cumulative release concentration of malic acid within 24 h was calculated. The results are as follows: Figure 5 As shown in (a), the cumulative release concentration of malic acid within 24 hours decreases with increasing iron tetrasulfide content in the hydrogel. This is because the added nano-metal sulfides can chelate with the hydroxyl and carboxyl groups on the polymer chain. With increasing metal sulfide content, more groups on the polymer chain are chelated, and the pore structure becomes more compact. Figure 4 The release of malic acid was limited by the PMQ-Fe3S4-a gel, resulting in a higher final malic acid release concentration compared to the other two gel groups, while the final malic acid release concentration of the PMQ-Fe3S4-c gel was lower than that of the other two gel groups. Furthermore, the sustained-release curves of malic acid showed that all three PMQ-Fe3S4 gels released malic acid over 72 hours, demonstrating a long-lasting sustained-release effect in buffer solution.

[0118] (2) The gels from Examples 4, 5, and 6 were placed in dialysis bags (MWCO = 100D) and incubated in phosphate buffer solution at pH 9.0 at 37°C. Three parallel groups were set up, and 2 mL of sample was taken from each group at regular intervals, with 2 mL of fresh buffer added. H2S in the solution was detected using methylene blue spectrophotometry. The sampled liquid was reacted with the prepared methylene blue solution, and the absorbance was measured at 665 nm. A curve was plotted with each sampling time point (0h, 0.5h, 1h, 2h, 4h, 6h, 8h, 12h, 16h, and 24h) as the x-axis and the cumulative percentage of H2S release at each sampling point as the y-axis. The results are shown below. Figure 5 As shown in (b), the sustained release rate of H2S increases rapidly in the first 6 hours, decreases after 6 hours, and stabilizes after 12 hours. Furthermore, the cumulative release concentration of H2S gradually increases with the increase of zinc sulfide content in the gel.

[0119] (3) The gels from Examples 4, 5, and 6 were placed in dialysis bags (MWCO = 500D) and then in phosphate buffer solution with pH = 9.0. Three parallel groups were set up and incubated at 37°C. The pH of the buffer solution was measured at regular intervals. A pH change curve was plotted with the sampling time (0h, 1h, 3h, 5h, 8h, 12h, 18h, 24h, 36h, 48h, 60h, 72h) as the x-axis and the pH at the corresponding time point as the y-axis. Figure 5 (c) As shown in the figure, the gel effectively lowers the pH of the alkaline buffer solution. With increasing gel release time, the pH of the buffer solution gradually decreases. However, with increasing iron(II,III) sulfide content in the gel, the pH change slows down. The buffer solution containing PMQ-Fe3S4-a gel decreases from pH 9.0 to 7.7, while the buffer solution containing PMQ-Fe3S4-c gel shows a more stable pH change, slowly decreasing from pH 9.1 to pH 7.9. This phenomenon is because as the metal sulfide content increases, the pores of the gel become denser, affecting the release of malic acid. Furthermore, some malic acid reacts with iron(II,III) sulfide to form weakly acidic hydrogen sulfide, thus resulting in a slower pH change in the buffer solution. Increasing the metal sulfide content in the gel can regulate the release of malic acid and avoid the heat generated by a violent acid-base neutralization reaction.

[0120] Figure 5 (a) The sustained-release curve of malic acid and Figure 5(b) The sustained-release curve of zinc sulfide demonstrates that the PMQ-Fe3S4 gel can release acid in stages and consume alkali over a long period. In the alkaline buffer solution, the gel releases malic acid and hydrogen sulfide in the first stage (0-24h) to remove alkali. During this stage, the gel continuously releases malic acid to consume alkali. When malic acid accumulates to a certain concentration, ferric sulfide in the gel reacts with it to generate hydrogen sulfide. This generates a penetrating weak acid gas to remove residual alkali in the deep corneal tissue and also regulates the concentration of malic acid, preventing excessively high malic acid concentration from causing a violent neutralization reaction with alkali and generating heat, thus avoiding secondary damage to the corneal wound. When ferric sulfide is gradually consumed completely, the gel no longer releases hydrogen sulfide, but the malic acid in the gel continues to be released to assist in removing alkali from the ocular surface. This is the second stage of acid release by the gel. In the corneal alkali burn model, the gel, through this graded acid release mechanism, can slowly and effectively neutralize residual alkali in the eye, effectively reducing the pH of the alkaline buffer solution (Figure a), and avoiding secondary damage to the wound caused by rapid acid-base reaction and heat generation.

[0121] Example 7

[0122] (1) Weigh 2g of polyvinyl alcohol (PVA) powder of type 2488 with an average molecular weight of 100,000 and passing through an 80-mesh sieve at room temperature, dissolve it in 50mL of water, and stir for 1h to obtain a PVA solution.

[0123] (2) Weigh 4g of citric acid (CA) and dissolve it in 20mL of water to obtain a citric acid solution. Take 5mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 1mL / min. Stir for 1h.

[0124] (3) Weigh 0.01g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 5.5mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 60℃ for 12h.

[0125] (4) Add the product from (3) to 80% ethanol and let it settle for 24 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 800D and dialyze for 2 days.

[0126] (5) The product in (4) was transferred to a 60°C oven and dried for 8 hours to obtain the grafted modified polyvinyl alcohol-citric acid ester (PVA-CA).

[0127] (6) Dissolve 2g of the product from (5) in 10mL of water and stir for 2h to obtain a PVA-CA solution.

[0128] (7) Weigh 0.5g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 1h to obtain quaternary ammonium salt chitosan solution. Take 4mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 30min to obtain PVA-CA / QCS solution.

[0129] (8) 0.006 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 8 min to obtain zinc sulfide colloidal solution.

[0130] (9) Under stirring, 0.7 mL of zinc sulfide colloidal solution (8) was slowly added dropwise to the solution (7) and stirred for 4 h. Then, it was transferred to -80℃ for freezing for 12 h and thawed at 25℃ for 8 h. A total of 3 freeze-thaw cycles were performed to obtain a graded acid-releasing, long-lasting alkali-consuming hydrogel PVA-CA / QCS-ZnS (PCQ-ZnS-a) loaded with zinc sulfide. The gel contained 0.42 mg of zinc sulfide nanoparticles.

[0131] Example 8

[0132] Unlike Example 7, in step (8), 0.008 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 8 min to obtain a zinc sulfide colloidal solution, and finally a graded acid-releasing, long-lasting alkali-consuming hydrogel PVA-CA / QCS-ZnS (PCQ-ZnS-b) loaded with zinc sulfide was obtained. This gel contains 0.56 mg of zinc sulfide nanoparticles.

[0133] Example 9

[0134] Unlike Example 7, in step (8), 0.01 g of zinc sulfide nanoparticles were vortexed in 10 mL of 50% ethanol aqueous solution for 8 min to obtain a zinc sulfide colloidal solution, and finally a graded acid-releasing, long-lasting alkali-consuming hydrogel PVA-CA / QCS-ZnS (PCQ-ZnS-c) loaded with zinc sulfide was obtained. This gel contains 0.70 mg of zinc sulfide nanoparticles.

[0135] Test Example 5

[0136] Unlike Test Example 4, the gels from Examples 7, 8, and 9 were placed in dialysis bags (MWCO = 100D) and in phosphate buffer solution at pH 9.0. Three parallel groups were set up, and the gels were incubated at 37°C. The citric acid release curve, hydrogen sulfide release curve, and pH change curve of the buffer solution for the gels from Examples 7, 8, and 9 were measured according to the operating procedures in Test Example 4. The results are as follows: Figure 6 As shown in (ac). From Figure 6As shown in Figure (a), the PCQ-ZnS gel continuously releases citric acid within 72 hours, with the highest release rate from 0 to 8 hours. The release rate decreases from 8 to 36 hours but remains stable. Furthermore, the cumulative release concentration of citric acid gradually decreases with increasing zinc sulfide content in the gel, consistent with the sustained-release curve of malic acid in Test Example 4. This further demonstrates that sulfides can slow down the release of organic acids by reacting with them. Figure (b) shows that the release rate of hydrogen sulfide is highest from 0 to 4 hours, flattens out from 5 to 24 hours, and the cumulative release concentration of hydrogen sulfide increases with increasing zinc sulfide content in the gel. Combining Figures (a) and (b), it can be demonstrated that the first stage of acid release from the gel consists of citric acid and hydrogen sulfide. After 24 hours, as sulfides are consumed, the release of hydrogen sulfide decreases, and the second stage of acid release is citric acid. Under this graded acid release mechanism, the gel can effectively lower the pH of the alkaline buffer. The pH change curve in Figure (c) shows that after 24 hours, PCQ-ZnS-a / b / c can lower the pH of the buffer from 9.0 to 7.5, 7.6, and 7.9, respectively. After h, the pH of the buffer solution containing the PCQ-ZnS-a / b / c gel decreased to 7.2, 7.4, and 7.7. Furthermore, the rate of pH change decreased with increasing zinc sulfide content. This is because the increased sulfide content leads to the consumption of more citric acid, thus reducing the rate of pH change. In practical applications of corneal alkali burns, an appropriate amount of sulfide is beneficial for regulating the release of organic acids, both by consuming the alkali solution to lower the pH and by avoiding secondary damage to the cornea caused by the heat generated from high-concentration acid-base reactions.

[0137] Comparative Example 3

[0138] (1) Weigh 3g of polyvinyl alcohol (PVA) powder of type 2488 with an average molecular weight of 120,000 and passing through a 100-mesh sieve at room temperature, dissolve it in 50mL of water, and stir for 1h to obtain a PVA solution.

[0139] (2) The product from (1) was added to 70% ethanol and precipitated for 48 hours, and then transferred to a dialysis bag with a molecular weight cutoff of 3000D for dialysis for 3 days.

[0140] (3) The product from (2) was transferred to a 60°C oven and dried for 8 hours to obtain purified polyvinyl alcohol (PVA).

[0141] (4) Dissolve 2g of the product from (3) in 30mL of water and stir for 3h to obtain a PVA solution.

[0142] (5) Weigh 2g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 2h to obtain quaternary ammonium salt chitosan solution. Take 3mL of quaternary ammonium salt chitosan solution and add it to the solution in (4) and stir for 30min.

[0143] (6) The product from (5) was transferred to -80℃ for 24 hours and thawed at 37℃ for 12 hours. The freeze-thaw cycle was repeated a total of 5 times to obtain PVA-QCS(PQ) hydrogel.

[0144] Comparative Example 4

[0145] (1) Weigh 3g of polyvinyl alcohol (PVA) powder of type 2488 with an average molecular weight of 120,000 and passing through a 100-mesh sieve at room temperature, dissolve it in 50mL of water, and stir for 1h to obtain a PVA solution.

[0146] (2) Weigh 4g of malic acid (MA) and dissolve it in 20mL of water to obtain a malic acid solution. Take 10mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 2mL / min. Stir for 2h.

[0147] (3) Weigh 0.1g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 6mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 70℃ for 24h.

[0148] (4) Add the product from (3) to 70% ethanol and let it settle for 48 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 3000D and dialyze for 3 days.

[0149] (5) The product in (4) was transferred to a 60°C oven and dried for 8 hours to obtain grafted modified polyvinyl alcohol-malate (PVA-MA).

[0150] (6) Dissolve 2g of the product from (5) in 30mL of water and stir for 3h to obtain a PVA-MA solution.

[0151] (7) Weigh 2g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 2h to obtain quaternary ammonium salt chitosan solution. Take 3mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 30min to obtain PVA-MA / QCS solution.

[0152] (8) The solution in (7) was transferred to -80℃ for freezing for 24 hours and thawed at 37℃ for 12 hours. A total of 5 freeze-thaw cycles were performed to obtain the hydrogel PVA-MA / QCS (PMQ).

[0153] Comparative Example 5

[0154] (1) Weigh 2g of polyvinyl alcohol (PVA) powder of type 2488 with an average molecular weight of 100,000 and passing through an 80-mesh sieve at room temperature, dissolve it in 50mL of water, and stir for 1h to obtain a PVA solution.

[0155] (2) Weigh 4g of citric acid (CA) and dissolve it in 20mL of water to obtain a citric acid solution. Take 5mL of this solution and add it dropwise to the PVA solution in (1) at a rate of 1mL / min. Stir for 1h.

[0156] (3) Weigh 0.01g of tin tetrachloride (SnCl4) and dissolve it in 5mL of water to obtain a tin tetrachloride solution. Take 5.5mL of the solution and add it to the solution in (2). Heat the solution in an oil bath at 60℃ for 12h.

[0157] (4) Add the product from (3) to 80% ethanol and let it settle for 24 hours. Then transfer it to a dialysis bag with a molecular weight cutoff of 800D and dialyze for 2 days.

[0158] (5) The product in (4) was transferred to a 60°C oven and dried for 8 hours to obtain the grafted modified polyvinyl alcohol-citric acid ester (PVA-CA).

[0159] (6) Dissolve 2g of the product from (5) in 10mL of water and stir for 2h to obtain a PVA-CA solution.

[0160] (7) Weigh 0.5g of quaternary ammonium salt chitosan and dissolve it in 10mL of water. Stir for 1h to obtain quaternary ammonium salt chitosan solution. Take 4mL of quaternary ammonium salt chitosan solution and add it to the solution in (6). Stir for 30min to obtain PVA-CA / QCS solution.

[0161] (8) The solution in (7) was transferred to -80℃ for freezing for 12h and 25℃ for 8h. The freeze-thaw cycle was repeated 3 times to obtain the hydrogel PVA-CA / QCS(PCQ).

[0162] Test Example 6

[0163] The therapeutic effects of PQ hydrogel (Comparative Example 3) without organic acid grafting and sulfide loading, PCQ hydrogel (Comparative Example 5) grafted only with citric acid, PMQ hydrogel (Comparative Example 4) grafted only with malic acid, PMQ-Fe3S4 hydrogel (Example 5) loaded with zinc sulfide and exhibiting graded acid release and long-lasting alkali consumption, and PCQ-ZnS hydrogel (Example 8) were determined using a mouse corneal alkali burn model. Sterile filter paper with a diameter of 3 mm was soaked in 1M NaOH solution. One piece was placed in the center of the right cornea of ​​a mouse for 30 seconds and then quickly removed. The eye was immediately rinsed with copious amounts of physiological saline for 1 minute. Sterile PQ hydrogel, PCQ hydrogel, PMQ hydrogel, PMQ-Fe3S4 hydrogel, and PCQ-ZnS hydrogel were then applied to the burned ocular surface, and the upper and lower eyelids were sutured. No other treatment was given to the burn wounds in the injured groups.

[0164] On days 0, 3, 7, and 14 post-surgery, a 10wt% sodium fluorescein solution was instilled onto the corneal surface for fluorescein staining. Changes in the eye were observed using a slit lamp on days 0, 3, 7, and 14 post-alkali burn. Figure 7As shown, in the damaged group, the cornea gradually became cloudy and opaque, and the corneal scar area gradually increased. By day 14, the cornea was completely opaque, and sodium fluorescein staining showed a large area of ​​positive results, indicating extensive corneal scarring accompanied by a large amount of neovascularization. In the PQ group, the corneal opacity was less than that in the damaged group. By day 14, the cornea showed a large amount of neovascularization and extensive scarring, and sodium fluorescein staining of the cornea showed a large area of ​​green fluorescence. This is because the quaternary ammonium salt chitosan in the gel has anti-inflammatory and antibacterial effects, which can improve the condition of corneal alkali burns. However, it cannot achieve a therapeutic effect. The corneas of the PMQ and PCQ groups showed significant therapeutic effects compared to the previous two groups. By day 14, the corneas became transparent, but there were still a small number of neovascularizations. Fluorescein staining showed large areas of diffuse spots. This is because the malic acid in the PMQ and PCQ gels can remove some of the residual alkali in the cornea and exert anti-inflammatory and antioxidant effects, which can significantly improve the corneal ulceration after alkali burns. However, malic acid or citric acid alone cannot remove the alkali in the deep tissues of the eye, so it cannot achieve a good therapeutic effect. PMQ-Fe3S4 gel and PCQ-ZnS hydrogel demonstrated excellent therapeutic effects. Although corneal opacity and scarring were still present on days 0 and 3 in both groups, the opacity and scarring tended to decrease. By day 7, corneal transparency increased and the scar area gradually decreased. By day 14, the cornea was nearly transparent, and sodium fluorescein staining was negative, indicating that no corneal scarring had occurred. This demonstrates that both PMQ-Fe3S4 hydrogel and PCQ-ZnS hydrogel have good therapeutic effects on alkali-burned corneas.

[0165] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a hydrogel that releases acid in stages and consumes alkali, characterized in that, Includes the following steps: (1) Add quaternary ammonium salt chitosan solution to polyvinyl alcohol-organic acid acid ester solution and stir evenly to obtain polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution; (2) Under stirring conditions, the nano metal sulfide colloidal solution was added to the polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution, stirred evenly, and repeatedly frozen and thawed. After thawing, a hydrogel that releases acid in stages and consumes alkali was obtained. The polyvinyl alcohol-organic acid ester compound in step (1) is prepared by the following method: A certain mass of polyvinyl alcohol is dissolved in water to obtain a polyvinyl alcohol solution. An organic acid solution is slowly added dropwise, and after stirring evenly, a tin tetrachloride catalyst solution is added. The mixture is heated in an oil bath at 40–90°C for 12–72 hours. After sedimentation, dialyzing, and drying, a polyvinyl alcohol-organic acid ester is obtained. The mass-to-volume ratio of polyvinyl alcohol to water (g / mL) is 1–3:10–50. The organic acid is at least one of malic acid, maleic acid, citric acid, and fumaric acid. The nano-metal sulfide is at least one of zinc sulfide, ferric tetrasulfide, ferrous sulfide, and manganese sulfide.

2. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 1, characterized in that, The organic acid solution is prepared by mixing organic acid and water at a mass-to-volume ratio of 0.5–4:5–20 g / mL.

3. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 1, characterized in that, In step (1), the mass-to-volume ratio of polyvinyl alcohol-organic acid ester to water in the polyvinyl alcohol-organic acid ester solution is 0.5-2.5:10-30 (g / mL). The mass-to-volume ratio of quaternary ammonium chitosan to water in the quaternary ammonium chitosan solution is 0.1–2.0:10–30 (g / mL). The volume ratio of the polyvinyl alcohol-organic acid ester solution to the quaternary ammonium salt chitosan solution is 5-15:1-5.

4. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 1, characterized in that, The volume ratio of the nano-metal sulfide colloidal solution and the polyvinyl alcohol-organic acid / quaternary ammonium salt chitosan solution in step (2) is 0.1-1:10-30.

5. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 1, characterized in that, The colloidal solution of nano-metal sulfide in step (2) is obtained by dispersing nano-metal sulfide particles in an ethanol-water mixture, with the mass-volume ratio of nano-metal sulfide, ethanol and water being (0.001~0.01)g:(5~10)mL:(5~10)mL.

6. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 1, characterized in that, The freezing temperature for repeated freeze-thaw cycles in step (2) is -20 to -80°C, the freezing time is 4 to 24 hours, and the number of freeze-thaw cycles is 2 to 5. The thawing temperature is 25–37°C, and the thawing time is 2–12 h.

7. The method for preparing a hydrogel that releases acid in stages and consumes alkali according to claim 5, characterized in that, The polyvinyl alcohol is at least one of type 1788, type 1792 and type 2488, and has a fineness of 80 to 100 mesh.

8. A hydrogel that releases acid in stages and consumes alkali, prepared by the method according to any one of claims 1 to 7.

9. The application of the hydrogel according to claim 8 in the preparation of alkali burn corneal wound materials.

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