Responsive drug release functional hydrogel and preparation method thereof

Abstract

The application discloses a functional hydrogel with responsive drug release and a preparation method thereof, and belongs to the technical field of biological medicines. The functional hydrogel matrix of the functional hydrogel with responsive drug release is a high-molecular functional hydrogel formed by physical crosslinking of polyvinyl alcohol and polyvinyl alcohol containing one or more responsive prodrugs through a freeze-thaw cycle. The carboxyl-containing drug is synthesized into a responsive prodrug through a two-step reaction, and then connected to the polyvinyl alcohol main chain. Finally, the functional hydrogel with responsive drug release is prepared through a freeze-thaw cycle method. The functional hydrogel with responsive drug release prepared by the application can be triggered to release drugs by active oxygen at a disease site, prolong the drug action time, improve the bioavailability of the drug, and be used for personalized treatment of diseases such as inflammation and infection.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a responsive drug-releasing functional hydrogel and its preparation method. Background Technology

[0002] Bacterial keratitis is one of the leading causes of corneal opacity and blindness. Timely and appropriate treatment is crucial for preventing complications such as corneal scarring, perforation, and even permanent visual impairment. The importance of treating corneal damage is equally self-evident, as it directly impacts a patient's vision and eye health. A common treatment for bacterial keratitis and corneal damage is the combined use of antibiotic and anti-inflammatory eye drops to control or prevent infection and reduce inflammation and pain. However, due to the poor retention of these eye drops, their bioavailability is extremely low, requiring frequent application. This not only leads to poor patient compliance but also causes significant fluctuations in drug concentration. High drug concentrations may result in unnecessary toxic side effects; while low concentrations may be insufficient to eradicate all bacteria, leading to drug resistance.

[0003] Functional hydrogels offer moisturizing benefits and prevent secondary damage from blinking, minimizing corneal stroma exposure. They also serve as drug delivery repositories, appropriately prolonging drug residence time on the ocular surface and maintaining therapeutic drug concentrations. However, this drug delivery method still presents challenges. Immersion-based drug delivery often results in high burst release rates, short release times, and significant fluctuations in drug concentration. Furthermore, preventing substantial leakage of the hydrogel during storage, leading to inaccurate dosage, remains a major challenge.

[0004] Reactive oxygen species (ROS) play a crucial role in inflammation, exacerbated at sites of infection and tissue damage. These ROS are important in regulating inflammatory responses and defending the host against invasive microorganisms; however, uncontrolled excesses of ROS can further damage host cells during wound healing. Inspired by this, ROS-responsive drug-release hydrogels utilize the elevated ROS environment characteristic of corneal injury and ocular surface inflammation to achieve adaptive drug release. They essentially "switch on" drug release at the injured or infected ocular surface. Compared to other ROS-responsive units, arylboronic esters selectively respond to H₂O₂, exhibiting excellent in vitro degradation kinetics under physiological conditions, stoichiometrically eliminating hydrogen peroxide and mimicking the action of catalase. More importantly, materials functionalized with arylboronic esters have shown good safety profiles in in vitro experiments and in vivo evaluations in various animal models. Therefore, developing such ROS-responsive drug delivery systems has enormous clinical application potential. This study uses phenylboronic acid, a reactive oxygen species, as a linking group. One end of the phenylboronic acid binds to the drug, while the other end forms a borate ester bond with the hydrogel polymer chain. Through coordination with boron atoms via H2O2, the BC bond is oxidized to form borate salts. In water, the boronic acid / ester and aromatic phenols are rapidly hydrolyzed, releasing active drug molecules simultaneously. This achieves the goal of on-demand drug release, that is, adjusting the drug release rate according to the severity of the disease, thereby releasing a higher dose in more severe cases. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, the present invention aims to provide a responsive drug-releasing functional hydrogel that can release drugs under the triggering of ROS on the inflamed ocular surface, achieving simultaneous and controllable release of different hydrophilic and hydrophobic drugs, prolonging the residence time, and significantly improving bioavailability.

[0006] Another objective of this invention is to provide a method for preparing polyvinyl alcohol containing a responsive prodrug.

[0007] Another object of the present invention is to provide the application of the above-mentioned polyvinyl alcohol containing a responsive prodrug.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a functional hydrogel with responsive drug release;

[0010] Furthermore, the responsive drug-releasing functional hydrogel matrix is ​​a high-molecular-weight functional hydrogel formed by physical cross-linking of polyvinyl alcohol chains initiated by a freeze-thaw cycle with polyvinyl alcohol containing one or more responsive prodrugs.

[0011] Furthermore, by weight percentage, the components account for the following percentages of the hydrogel: polyvinyl alcohol accounts for 0% to 90% of the dry weight of the matrix, polyvinyl alcohol containing one or more responsive prodrugs accounts for 10% to 100% of the dry weight of the matrix, and the total is 100%.

[0012] Further, the polyvinyl alcohol containing one or more responsive prodrugs has a drug component accounting for 0.01% to 30% of the matrix by weight; preferably, the drug component accounts for 1% to 5% of the matrix by weight.

[0013] The method for preparing the responsive drug-releasing functional hydrogel includes the following steps:

[0014] Polyvinyl alcohol (PVA) and PVA containing one or more responsive prodrugs are placed in a mixed solvent of dimethyl sulfoxide (DMSO) and water. After dissolving by heating and stirring, the mixture is poured into a mold and frozen at -20°C, followed by thawing at room temperature. This freeze-thaw cycle is repeated. The DMSO is washed away with deionized water to obtain a drug-loaded functional hydrogel with responsive drug release. Preferably, the freeze-thaw cycle is repeated 4 times: freezing at -20°C for 7 hours, followed by thawing at room temperature for 3 hours.

[0015] The polyvinyl alcohol containing one or more responsive prodrugs has the following general chemical formula:

[0016]

[0017] Wherein, R1 is one or more combinations of carboxyl-containing drugs, linked to the benzene ring via an ester group. R2 indicates that the hydrogen atoms on the benzene ring can be replaced by any group. The degree of polymerization of polyvinyl alcohol is 1000–10000, preferably 1500–2000. m≥1.

[0018] The present invention provides a method for preparing polyvinyl alcohol containing one or more responsive prodrugs, comprising the following steps:

[0019] (1) Esterification of a carboxyl-containing drug, hydroxymethylphenylboronic acid pinacol ester or its derivative in an organic solvent in the presence of a catalyst to obtain an intermediate compound, denoted as Drug-PBE;

[0020] (2) The intermediate compound Drug-PBE was reacted with sodium periodate to remove pinacol, and the intermediate compound was obtained, denoted as Drug-PBA.

[0021] (3) The intermediate compound Drug-PBA is esterified with the diol structure of polyvinyl alcohol to generate polyvinyl alcohol containing one or more responsive prodrugs.

[0022] Further, the intermediate compound Drug-PBE described in step (1) is prepared as follows: a carboxyl-containing drug, pinacol hydroxymethylphenylboronic acid or its derivative, and 4-dimethylaminopyridine are dissolved in an organic solvent and stirred at room temperature for 10-15 min; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added and stirred at room temperature for more than 12 hours in the dark; after the reaction is completed, the product Drug-PBE is obtained by column chromatography separation and post-processing.

[0023] The molar ratio of the carboxyl-containing drug, pinacol hydroxymethylphenylboronic acid or its derivative, 4-dimethylaminopyridine, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (1-1.2):(1-1.2):(1-1.2):(1.2-1.5); the organic solvent is one of dichloromethane, acetonitrile, and acetone; the pinacol hydroxymethylphenylboronic acid or its derivative is preferably pinacol 4-hydroxymethylphenylboronic acid.

[0024] Further, the preparation method of the intermediate compound Drug-PBA in step (2) is as follows: the intermediate compound Drug-PBE, sodium periodate and ammonium acetate are dissolved in a solvent, stirred at room temperature for more than 12 hours, and after the reaction is completed, the product Drug-PBA is obtained by separation and processing by column chromatography.

[0025] The molar ratio of Drug-PBE, sodium periodate, and ammonium acetate is (1-3):(1-3):(1-3); the solvent is a mixture of acetone and water.

[0026] Further, the preparation method of polyvinyl alcohol containing one or more responsive prodrugs in step (3) is as follows: polyvinyl alcohol and dimethyl sulfoxide are added to a flask and heated to 90°C with stirring until completely dissolved. After dissolution, the mixture is cooled to room temperature, intermediate compound Drug-PBA and organic base are added, the mixture is stirred overnight at room temperature, and the product is dialyzed in deionized water for 24-48 hours. After drying, polyvinyl alcohol containing one or more responsive prodrugs is obtained.

[0027] The molar ratio of Drug-PBA, polyvinyl alcohol, and organic base is 1:(1-20):(1-10); the organic base is one of triethylamine, trimethylamine, diisopropylethylamine, DBU, and pyridine.

[0028] Furthermore, the carboxyl-containing drug is one or more of drugs containing a carboxyl group or modified drugs containing a carboxyl group, preferably nonsteroidal anti-inflammatory drugs and antibacterial drugs, and more preferably diclofenac or levofloxacin.

[0029] Furthermore, the polyvinyl alcohol containing one or more responsive prodrugs is loaded with only one drug per polyvinyl alcohol molecule, and after mixing, polyvinyl alcohol containing multiple drugs is obtained, or a polyvinyl alcohol molecule is simultaneously loaded with multiple drugs.

[0030] This invention provides the application of the responsive drug-releasing functional hydrogel in the preparation of medical devices for treating bacterial keratitis.

[0031] The functional hydrogel with responsive drug release and its preparation method of the present invention have the following advantages:

[0032] (1) The functional hydrogel loaded with reactive oxygen species responsive prodrug designed and synthesized in this invention has adjustable drug dosage and controllable drug release, which effectively increases the drug retention time and bioavailability in the eye.

[0033] (2) The drug-loaded functional hydrogel is prepared by physical cross-linking of polyvinyl alcohol, namely the freeze-thaw cycle method. The preparation process is simple, does not contain organic cross-linking agents, has high light transmittance and good mechanical properties.

[0034] (3) Almost no drug leakage occurs during storage, giving full play to the role of the disease response "switch" to achieve effective prevention and treatment of ocular surface bacterial infection and inflammation.

[0035] (4) It is widely applicable to the development of inflammatory responsive prodrugs of various drugs containing carboxyl groups or modified drugs that can carry carboxyl groups and other similar compounds, and provides ideas for responsive drug release at other inflammatory sites.

[0036] (5) The responsive drug-releasing functional hydrogel prepared by the present invention can be triggered by reactive oxygen species at the site of ocular surface disease to release drugs, prolong the drug action time, improve the ocular bioavailability of drugs, and be used for personalized treatment of ocular surface inflammation or infection and other diseases.

[0037] (6) In this invention, the drug is chemically bonded within the polymer hydrogel rather than simply infiltrated. Chemical bonding allows for more precise control of the drug release rate and duration; it also generally means a tighter and more stable bond between the drug and the hydrogel. This stability protects the drug from external conditions (such as light, humidity, or temperature changes), prolonging its effective period and enhancing its stability. Chemical bonding also promotes efficient drug delivery in the body, as the drug is fixed to the target area by the polymer hydrogel, avoiding rapid release or loss that might occur with simple infiltration. Precise control of the drug release rate and location reduces side effects caused by drugs in non-target areas; this targeted release reduces the impact on the overall body system, thereby improving the safety and effectiveness of treatment. By fixing drug molecules to the polymer hydrogel through chemical bonds, the release process becomes more persistent and stable. This persistent release reduces the frequency of drug administration during treatment, improving patient convenience and compliance. Attached Figure Description

[0038] Figure 1 The proton NMR spectrum and mass spectrum of diclofenac-PBE provided by this invention.

[0039] Figure 2 The proton NMR spectrum and mass spectrum of diclofenac-PBA provided by this invention.

[0040] Figure 3 The proton NMR spectrum and mass spectrum of levofloxacin-PBE provided by this invention.

[0041] Figure 4 The proton NMR spectrum and mass spectrum of levofloxacin-PBA provided by this invention.

[0042] Figure 5 The images show the appearance and transmittance of the self-made blank hydrogel and responsive drug-release hydrogel provided by this invention.

[0043] Figure 6 The graph shows the water content determination results of the self-made blank hydrogel, the responsive drug-releasing hydrogel, and the commercially available contact lens provided by this invention.

[0044] Figure 7 The mechanical strength test results of the self-made blank hydrogel, the responsive drug-releasing hydrogel, and the commercially available contact lens provided by this invention are shown in the figure.

[0045] Figure 8 The in vitro release results of the responsive drug-releasing hydrogel provided by the present invention are shown in the figure.

[0046] Figure 9 The image provided by this invention shows the results of determining biofilm formation using the crystal violet staining method.

[0047] Figure 10 The image shows the results of biofilm formation determination using plate counting, as provided by this invention.

[0048] Figure 11 The image provided by this invention shows the results of observing biofilm formation using a scanning electron microscope.

[0049] Figure 12 The clinical scoring results of the efficacy experiment for bacterial keratitis provided by this invention are shown in the figure.

[0050] Figure 13 The image shows the results of tissue sections and corneal thickness measurements in the bacterial keratitis efficacy experiment provided by this invention. Detailed Implementation

[0051] This invention relates to a responsive drug-releasing functional hydrogel, wherein the drug-loaded functional hydrogel is a high-molecular-weight functional hydrogel formed by physical cross-linking of polyvinyl alcohol and polyvinyl alcohol containing one or more responsive prodrugs through a freeze-thaw cycle.

[0052] The polyvinyl alcohol containing one or more responsive prodrugs is synthesized through a two-step reaction. Each polyvinyl alcohol molecule may be loaded with only one drug, and the mixture yields polyvinyl alcohol containing multiple drugs; alternatively, a single polyvinyl alcohol molecule may be co-loaded with multiple drugs. The loaded drugs may be drugs with carboxyl groups or modified drugs with carboxyl groups.

[0053] Transmittance measurement:

[0054] A fully hydrated contact lens in simulated tear fluid (STF) was wiped dry and placed on the inner surface of a quartz tube. Transmittance was measured using a UV-Vis spectrophotometer with a wavelength range of 400–800 nm.

[0055] Moisture content measurement:

[0056] The polymerized functional hydrogel was left to stand at room temperature in a dry state for 24 hours, and the mass W1 of the functional hydrogel was measured. The dried functional hydrogel was then placed in simulated tear fluid for equilibration for 24 hours, and the weight W2 of the functional hydrogel was measured. The water content of each functional hydrogel was calculated as follows: Water content = (W2 – W1) / W1 × 100%.

[0057] Mechanical strength test:

[0058] The fully hydrated functional hydrogel was placed in a force gauge fixture, and the fixture was slowly stretched at a constant speed. The breaking tension and length before and after the test were recorded, and the tensile strength and elongation at break were calculated using the following formulas: Tensile strength (δ) = F / (a·b); Elongation at break E% = (L2-L1) / L1×100% where F is the breaking tension, a is the diameter of the functional hydrogel, b is the thickness of the functional hydrogel, L1 is the length before stretching, and L2 is the breaking length.

[0059] Response in vitro release assay:

[0060] In vitro release was performed in 24-well plates. 1 mL of STF containing 0, 50, or 100 μM H₂O₂ was added to each well. The drug-loaded functional hydrogel was placed in the release medium, and the 24-well plate was placed in a water bath at 35°C and shaken at 50 rpm. At regular intervals, 1 mL of release medium was removed, the drug concentration was measured, and 1 mL of fresh release medium was added simultaneously. Furthermore, the release medium (100 μM H₂O₂ or STF) was replaced every 12 hours to investigate the responsive drug release characteristics of the functional hydrogel.

[0061] The following detailed description of a responsive drug-releasing functional hydrogel and its preparation method according to the present invention, with reference to specific embodiments, is provided. The technical solution of the present invention includes, but is not limited to, the following embodiments.

[0062] Example 1: Preparation of a functional hydrogel loaded with a diclofenac responsive prodrug

[0063] (1) Preparation of the intermediate compound diclofenac-PBE

[0064] Diclofenac (1.2 equivalents), pinacol 4-hydroxymethylphenylboronic acid (1 equivalent), and 4-dimethylaminopyridine (1.2 equivalents) were completely dissolved in dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 equivalents) was added to the mixture. The reaction mixture was stirred overnight at room temperature. The product was purified by column chromatography with n-hexane-ethyl acetate (10:1, v / v) to give the oily product diclofenac-PBE. The 1H NMR and mass spectrometry results are shown in the appendix. Figure 1 .

[0065] (2) Preparation of the intermediate compound diclofenac-PBA

[0066] Diclofenac-PBE (1 equivalent), sodium periodate (3 equivalents), and ammonium acetate (3 equivalents) were dissolved in a mixture of acetone and water (2:1, v / v) in a flask and stirred overnight at room temperature. After removing acetone by rotary evaporation under reduced pressure, the product was dissolved in ethyl acetate and washed with saturated brine. The upper ethyl acetate layer was separated and purified by dichloromethane-methanol (50:1, v / v) column chromatography to obtain a white powder, diclofenac-PBA. The 1H NMR and mass spectrometry results are shown in the appendix. Figure 2 .

[0067] (3) Preparation of polyvinyl alcohol containing diclofenac responsive prodrug

[0068] PVA (400 mg) and DMSO (10 mL) were added to a flask and heated to 90 °C with stirring until completely dissolved. After dissolution, the mixture was cooled to room temperature, and diclofenac-PBA (100 mg) and triethylamine (100 mg) were added. The mixture was stirred overnight at room temperature. The product was then dialyzed against deionized water for 24 hours. The product was dried at 50 °C to obtain a diclofenac-polyvinyl alcohol transparent film.

[0069] (4) Preparation of functional hydrogels containing diclofenac responsive prodrugs

[0070] 200 mg of polyvinyl alcohol and 40 mg of diclofenac-polyvinyl alcohol were placed in a mixture of 800 μL DMSO and 200 μL water. The mixture was stirred at 90 °C until dissolved. After cooling to 60 °C, 60 mg / tablet was poured into a mold and frozen at -20 °C for 7 hours, then thawed at room temperature for 3 hours. This freeze-thaw cycle was repeated 4 times. After rinsing with deionized water to remove DMSO, a functional hydrogel loaded with a diclofenac responsive prodrug was obtained.

[0071] Example 2: Preparation of a functional hydrogel loaded with a levofloxacin responsive prodrug

[0072] (1) Preparation of the intermediate compound levofloxacin-PBE

[0073] Levofloxacin (1.2 equivalents), pinacol 4-hydroxymethylphenylboronic acid (1 equivalent), and 4-dimethylaminopyridine (1.2 equivalents) were completely dissolved in dichloromethane. Then, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (1.2 equivalents) was added to the mixture. The reaction mixture was stirred overnight at room temperature. The product was separated and purified by preparative liquid chromatography to obtain a light yellow powder, levofloxacin-PBE. The 1H NMR and mass spectrometry results are shown in the appendix. Figure 3 .

[0074] (2) Preparation of the intermediate compound levofloxacin-PBA

[0075] Levofloxacin-PBE (1 equivalent), sodium periodate (3 equivalents), and ammonium acetate (3 equivalents) were dissolved in a mixture of acetone and water (2:1, v / v) in a flask and stirred overnight at room temperature. After removing acetone by rotary evaporation under reduced pressure, the product was dissolved in ethyl acetate and washed with saturated brine. The upper ethyl acetate layer was separated and purified by column chromatography to obtain a light yellow powder of levofloxacin-PBA. The 1H NMR and mass spectrometry results are shown in the appendix. Figure 4 .

[0076] (3) Preparation of polyvinyl alcohol containing levofloxacin responsive prodrug

[0077] PVA (400 mg) and DMSO (10 mL) were added to a flask and heated to 90 °C with stirring until completely dissolved. After dissolution, the mixture was cooled to room temperature, and levofloxacin-PBA (100 mg) and triethylamine (100 mg) were added. The mixture was stirred overnight at room temperature. The product was then dialyzed against deionized water for 24 hours. The product was dried at 50 °C to obtain a levofloxacin-polyvinyl alcohol transparent film.

[0078] (4) Preparation of functional hydrogels containing levofloxacin responsive prodrug

[0079] 200 mg of polyvinyl alcohol and 40 mg of levofloxacin-polyvinyl alcohol were placed in a mixture of 800 μL of DMSO and 200 μL of water. The mixture was stirred at 90 °C until dissolved. After cooling to 60 °C, 60 mg / tablet was poured into a mold and frozen at -20 °C for 7 hours, then thawed at room temperature for 3 hours. This freeze-thaw cycle was repeated 4 times. The gel was then rinsed with deionized water to remove DMSO, yielding a functional hydrogel loaded with a levofloxacin-responsive prodrug.

[0080] Example 3: Preparation of a functional hydrogel loaded with levofloxacin and diclofenac responsive prodrugs

[0081] (1) Diclofenac-PBA and levofloxacin-PBA were prepared in accordance with the same method as in Example 1 and Example 2.

[0082] (2) Preparation of polyvinyl alcohol containing levofloxacin and diclofenac responsive prodrugs

[0083] PVA (340 mg) and DMSO (10 mL) were added to a flask and heated to 90 °C with stirring until completely dissolved. After dissolution, the mixture was cooled to room temperature, and diclofenac-PBA (40 mg), levofloxacin-PBA (120 mg), and triethylamine (200 mg) were added. The mixture was stirred overnight at room temperature. The product was then dialyzed against deionized water for 24 hours. The product was dried at 50 °C to obtain a levofloxacin & diclofenac-polyvinyl alcohol transparent film.

[0084] (3) Preparation of functional hydrogels containing levofloxacin and diclofenac responsive prodrugs

[0085] 200 mg of polyvinyl alcohol and 40 mg of levofloxacin & diclofenac-polyvinyl alcohol were placed in a mixture of 800 μL DMSO and 200 μL water. The mixture was stirred at 90 °C until dissolved. After cooling to 60 °C, 60 mg / tablet was poured into a mold and frozen at -20 °C for 7 hours, then thawed at room temperature for 3 hours. This freeze-thaw cycle was repeated 4 times. The gel was then rinsed with deionized water to remove DMSO, yielding a functional hydrogel loaded with a levofloxacin responsive prodrug.

[0086] Example 4: Physical property characterization

[0087] The drug-loaded functional hydrogel prepared in Example 3 and a blank polyvinyl alcohol contact lens were used, and the commercially available polyvinyl alcohol contact lens AquaComfort was used. (Alcon Inc.) was used as a control; the results of measuring several physical properties of the contact lens, including light transmittance, water content, and mechanical properties, are attached. Figures 5-7 Therefore, the prepared drug-loaded functional hydrogel and blank polyvinyl alcohol contact lens both have a light transmittance of over 90%, a moderate water content (65%–75%) similar to commercially available contact lenses, and significantly stronger mechanical strength than commercially available contact lenses.

[0088] Example 5: In vitro release

[0089] The in vitro drug release of levofloxacin and diclofenac from the functional hydrogel samples prepared in Example 3 was investigated in simulated tears containing 0, 50, and 100 μM H2O2. The results are attached. Figure 8 As shown, both levofloxacin and diclofenac can regulate their drug release rates according to the concentration of H2O2. Specifically, higher H2O2 concentrations result in faster drug release, exhibiting a relatively stable zero-order release. Furthermore, this functional hydrogel can release the drug in the presence of H2O2 and rapidly cease drug release in the absence of H2O2, demonstrating an intelligent drug release pattern. This drug release characteristic allows the functional hydrogel to flexibly adjust the drug release rate according to different disease states, maximizing drug utilization while minimizing unnecessary side effects.

[0090] Example 6: Antibacterial and anti-biofilm properties

[0091] The antibacterial and antibiofilm properties of the drug-loaded functional hydrogel prepared in Example 3 and a blank polyvinyl alcohol hydrogel were studied. 2 mL of Staphylococcus aureus bacterial suspension (approximately 10...) was added... 6CFU / mL was added to the wells of a 12-well plate. Blank polyvinyl alcohol hydrogel, blank polyvinyl alcohol hydrogel + 100 μM H2O2, responsive drug-releasing functional hydrogel, and responsive drug-releasing functional hydrogel + 100 μM H2O2 were added, and the plate was incubated at 37°C for 24 hours to form a biofilm. The biofilm was analyzed using crystal violet staining. Bacteria attached to the functional hydrogel were counted. The hydrogel was placed in sterile water to gently rinse away weakly attached bacteria. The hydrogel was then placed in a sterile EP tube containing 3 mL of sterile water and vortexed vigorously for 2 minutes. Bacterial counts were performed using serial dilutions and plate counting. Results are shown in the attached figure. Figure 9 and Figure 10 Regardless of whether H2O2 is added, the functional hydrogels with responsive drug release have a good ability to prevent biofilm formation, while the blank hydrogels without drugs do not have this property.

[0092] In addition, the hydrogel was gently washed with sterile water and fixed in 1 mL of 2.5% glutaraldehyde for 30 minutes. After rinsing three times with sterile water, 2 mL of methanol was added for dehydration for 30 minutes. The methanol was removed and the lens was dried for SEM observation. (See attached image) Figure 11 As shown, blank hydrogels cannot resist bacterial deposition on their surface, while responsive drug-releasing functional hydrogels remain smooth without deposition. If bacteria form a biofilm on the hydrogel surface, even if the initial infection focus is treated and cleared, the bacteria within the biofilm may reinfect the eye, leading to recurrent infections. Therefore, the functional hydrogel prepared in this invention can reduce the risk of infection recurrence, maintain the hydrogel's breathability and hygiene, and improve overall wearing comfort when used as an ocular surface insert.

[0093] Example 7: Evaluation of the efficacy of drugs for bacterial keratitis

[0094] A rabbit model of bacterial keratitis was established by injecting bacterial solution into the corneal stroma. Twenty-four hours after inoculation, 18 rabbits were randomly divided into six groups: PBS eye drops, levofloxacin eye drops, diclofenac eye drops, levofloxacin + diclofenac eye drops, blank hydrogel, and responsive drug-release hydrogel. The eye drops were administered at 50 μL three times daily; the hydrogel group received the drops every 5 days for a total of 15 days. Figure 12 As shown, the corneal infection in the model group worsened within 15 days, manifesting as corneal edema, opacity, and purulent exudate from the anterior chamber. Corneal perforation occurred, accompanied by significant neovascularization. Due to the adaptive release of the responsive hydrogel, the corneal infection was controlled more quickly, comparable to the effect of combined diclofenac and levofloxacin eye drops. Results from pathological tissue sections and corneal thickness measurements are attached. Figure 13As shown in the figure, the control group and the blank hydrogel group showed abnormal corneal structure, significant infiltration of inflammatory cells, and significant corneal thickening. After responsive hydrogel treatment, the corneal thickness returned to a normal level, only about 500 μm, and its effect was comparable to that of frequent and repeated use of diclofenac and levofloxacin eye drops.

Claims

1. A responsive drug-releasing functional hydrogel, characterized in that, The responsive drug-releasing functional hydrogel matrix is ​​polyvinyl alcohol and polyvinyl alcohol containing levofloxacin and diclofenac responsive prodrugs, and is a high-molecular functional hydrogel formed by physical cross-linking of polyvinyl alcohol chains through a freeze-thaw cycle. The polyvinyl alcohol containing levofloxacin and diclofenac responsive prodrugs is prepared by the following method: Diclofenac or levofloxacin, 4-hydroxymethylphenylboronic acid pinacol ester, and a catalyst are esterified in an organic solvent to obtain diclofenac-PBE or levofloxacin-PBE; diclofenac-PBE or levofloxacin-PBE is then reacted with sodium periodate to remove pinacol to obtain diclofenac-PBA or levofloxacin-PBA. The prepared diclofenac-PBA and levofloxacin-PBA were esterified with the diol structure of polyvinyl alcohol to obtain polyvinyl alcohol containing levofloxacin and diclofenac responsive prodrugs. The chemical structures of diclofenac-PBA or levofloxacin-PBA are represented as follows: ； R1 is diclofenac or levofloxacin, which is linked to the benzene ring via an ester group; R2 indicates that the hydrogen atoms on the benzene ring can be replaced by any group; the degree of polymerization of polyvinyl alcohol is 1000 to 10000; m ≥ 1.

2. A method for preparing the responsive drug-releasing functional hydrogel according to claim 1, characterized in that, The process includes the following steps: Polyvinyl alcohol (PVA) and DMSO are added to a reaction flask, stirred and heated until completely dissolved. After dissolution, the mixture is cooled to room temperature. Diclofenac-PBA, levofloxacin-PBA, and triethylamine are added, and the mixture is stirred and reacted at room temperature. After the reaction is complete, the product is dialyzed against deionized water and dried to obtain PVA containing levofloxacin and diclofenac responsive prodrugs. PVA and the PVA containing levofloxacin and diclofenac responsive prodrugs are placed in a mixed solvent of DMSO and water. After heating and stirring to dissolve, the mixture is poured into a mold and frozen at -20°C. It is then thawed at room temperature, and this freeze-thaw cycle is repeated four times. DMSO is washed away with deionized water to obtain a functional hydrogel loaded with levofloxacin and diclofenac responsive prodrugs. The preparation methods for diclofenac-PBA and levofloxacin-PBA are as follows: Diclofenac or levofloxacin, 4-hydroxymethylphenylboronic acid pinacol ester, and 4-dimethylaminopyridine are dissolved in an organic solvent and stirred at room temperature; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is added, and the mixture is stirred at room temperature under light-protected conditions until the reaction is complete. After separation and processing by column chromatography, diclofenac-PBE or levofloxacin-PBE is obtained; or diclofenac-PBE or levofloxacin-PBE, sodium periodate, and ammonium acetate are dissolved in a solvent and stirred at room temperature until the reaction is complete. After separation and processing by column chromatography, diclofenac-PBA or levofloxacin-PBA is obtained.

3. The method for preparing the responsive drug-releasing functional hydrogel according to claim 2, characterized in that, The molar ratio of diclofenac or levofloxacin, 4-hydroxymethylphenylboronic acid pinacol ester, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is (1~1.2):(1~1.2):(1~1.2):(1.2~1.5); the organic solvent is one of dichloromethane, acetonitrile and acetone.

4. The method for preparing the responsive drug-releasing functional hydrogel according to claim 2, characterized in that, The molar ratio of diclofenac-PBE or levofloxacin-PBE, sodium periodate and ammonium acetate is (1~3): (1~3): (1~3); the solvent is a mixture of acetone and water.

5. The use of the responsive drug-releasing functional hydrogel of claim 1 in the preparation of a medicament for treating bacterial keratitis.

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