A fast photo-crosslinking drug-releasing multifunctional hydrogel contact lens and a preparation method thereof

Abstract

The application discloses a kind of quick photo-crosslinking drug release type multifunctional hydrogel corneal contact lens and its preparation method, the preparation method includes the following steps: (1) according to proportion, quaternary ammonium chitosan, acrylate monomer, methacrylic acid, crosslinking agent and initiator are made into reaction mixture solution;(2) the reaction mixture solution is transferred to mould, and free radical polymerization is carried out under ultraviolet light, after reaction ends, the hydrogel is removed and washed after demolding, and freeze-drying treatment is carried out, to obtain freeze-dried hydrogel;(3) the freeze-dried hydrogel is immersed in drug monomer solution until the hydrogel is completely swollen.The corneal contact lens prepared by the method of the application can be polymerized in one step, quickly prepared, the water content of finished product is suitable, has excellent light transmittance and mechanical properties, can effectively load traditional Chinese medicine antibacterial drug, and prolongs the release time of drug, and has remarkable curative effect in the treatment of bacterial keratitis, and as therapeutic corneal contact lens has wide market application prospect.

Description

Technical Field

[0001] This invention relates to the field of polymer hydrogel technology, specifically to a fast photocrosslinked drug-releasing multifunctional hydrogel corneal contact lens and its preparation method. Background Technology

[0002] Currently, eye drops containing broad-spectrum antibiotics are commonly used in clinical practice to treat BK. However, due to antibiotic resistance caused by overuse and the limitations of traditional administration methods, there is an urgent need to develop more effective treatments for BK.

[0003] Drug-eluting contact lenses, by maintaining a more constant and controllable drug concentration in the tear film to provide higher drug bioavailability and fewer side effects, have become a current research hotspot in ophthalmic treatment. When a drug-eluting contact lens is worn, most of the loaded drug diffuses from the lens into the posterior tear film. Protected by the lens, the released drug prevents blinking reflexes and tear exchange, has a longer pre-corneal residence time, and a more direct diffusion path to the ocular surface. Meanwhile, to further improve the function of corneal contact lenses, researchers have begun to focus on strategies to achieve drug loading and performance optimization by introducing other monomers. Acrylic monomers are currently the main materials for contact lenses on the market, but they still have some problems, such as being prone to bacterial adhesion and not suitable for prolonged wear, which limits their application range. There is an urgent need to develop a contact lens that improves its insufficient antibacterial properties.

[0004] Acrylic monomers contain hydrophilic hydroxyl groups on their side chains, which not only endows the materials with good hydrophilicity but also allows for chemical modification, providing a solid foundation for the functional modification of corneal contact lenses.

[0005] Chitosan (CS) is a chitin-derived biopolymer with high biocompatibility, non-toxicity, and good biodegradability. However, its high crystallinity and low solubility limit its application in the biomedical field. To overcome these limitations, researchers have developed quaternized chitosan (QCS), which shows great potential in biomedical applications due to its good water solubility, biocompatibility, biodegradability, and antibacterial activity, as well as carrying a large number of cationic charges.

[0006] Traditional Chinese medicine (TCM) ingredients have received increasing attention in the ophthalmology field in recent years due to their safety, effectiveness, low toxicity, and lack of drug resistance compared to antibiotics. Currently, there are still many gaps in research on the application of TCM ingredients in ophthalmic medical materials. For example, TCM polyphenols and flavonoids often possess anti-inflammatory, antioxidant, and antibacterial properties, and their abundant active groups provide a solid foundation for the innovative modification and functionalization of ophthalmic materials.

[0007] While the aforementioned materials exhibit unique advantages in individual applications, their limited application in ophthalmic treatments is hampered by challenges such as single-performance limitations and insufficient bioavailability resulting from traditional administration methods. Addressing the limitations of single materials in ophthalmic therapy necessitates the development of innovative material combination strategies. This study aims to develop a novel ophthalmic formulation by integrating the chemical plasticity of acrylate monomers, the antibacterial properties of quaternized chitosan, and the multi-target, multi-pathway therapeutic advantages of traditional Chinese medicine ingredients. This combination strategy is expected to not only enhance the overall performance of the materials but also incorporate multiple bioactivities to achieve more effective and sustained therapeutic effects. Compared to traditional single-drug eye drops, this combination formulation will reduce the frequency of administration, decrease eye irritation, improve patient compliance, and thus significantly enhance the bioavailability of ophthalmic drugs. This innovative combination formulation holds promise for filling a gap in ophthalmic therapy, demonstrating significant application potential and clinical value.

[0008] Furthermore, the fabrication of traditional contact lenses often requires high temperatures and long processing times, resulting in low efficiency and complex processes that are unsuitable for industrial production. Therefore, there is an urgent need to develop a simple and efficient new fabrication method. Photocrosslinking technology, which utilizes specific wavelengths of light (such as ultraviolet or visible light) to trigger a reaction and promote the formation of stable covalent bonds between molecules, is gradually becoming a powerful tool for solving this problem. Specifically, the photocrosslinking process involves the use of a photoinitiator, which is activated under light conditions to initiate a polymerization reaction of double bonds in monomers or polymers, thereby rapidly forming a stable network structure. This technology offers mild reaction conditions, rapid fabrication speed, and excellent biocompatibility, making it a simpler and more efficient fabrication process compared to traditional thermal polymerization crosslinking methods. The application of photocrosslinking technology is expected to significantly improve the production efficiency of contact lenses, simplify manufacturing processes, and open up new possibilities for large-scale industrial production. Summary of the Invention

[0009] The purpose of this invention is to provide a rapid photocrosslinking drug-releasing multifunctional hydrogel corneal contact lens and its preparation method, overcoming the technical problems of complex manufacturing processes, insufficient antibacterial properties of materials, and safety concerns regarding various initiators in traditional corneal contact lenses. The drug-releasing multifunctional hydrogel corneal contact lens provided by this invention can be polymerized in one step and rapidly prepared. The finished product has suitable water content, excellent light transmittance, and superior mechanical properties. It can effectively load traditional Chinese medicine antibacterial drugs and prolong the drug release time, demonstrating excellent efficacy in the treatment of bacterial keratitis. As a therapeutic corneal contact lens, it has broad market application prospects.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] In a first aspect, the present invention claims protection for a method for preparing a fast photocrosslinked drug-releasing multifunctional hydrogel corneal contact lens, the method comprising the following steps:

[0012] (1) Add quaternized chitosan, acrylate monomers and methacrylic acid to water in proportion, and use ultrasound and stirring to completely dissolve the quaternized chitosan; then add crosslinking agent and initiator in sequence, and use ultrasound and stirring to completely dissolve the initiator to form a reaction mixture;

[0013] (2) Transfer the reaction mixture into a mold and carry out a free radical polymerization reaction under ultraviolet light. After the reaction is completed, demold the hydrogel and wash it. Freeze-dry the washed hydrogel to obtain a freeze-dried hydrogel.

[0014] (3) Immerse the freeze-dried hydrogel in the drug monomer solution until the hydrogel is completely swollen to obtain a drug-release multifunctional hydrogel corneal contact lens.

[0015] Further, the content of each component in the reaction mixture in step (1) is as follows: acrylate monomers 30 vol%–80 vol%, methacrylic acid 0.6 vol%–2 vol%, quaternized chitosan 0.001–3 g / L, crosslinking agent 0.5 vol%–1.6 vol%, initiator 0.01–1.5 g / L, and the balance being water. Preferably, the content of each component in the reaction mixture is as follows: acrylate monomers 40 vol%–80 vol%, methacrylic acid 0.6 vol%–2 vol%, quaternized chitosan 0.1–3 g / L, crosslinking agent 0.5 vol%–1.6 vol%, initiator 0.1–1.5 g / L, and the balance being water. More preferably, the content of each component in the reaction mixture is as follows: 50 vol% to 80 vol% acrylate monomers, 0.6 vol% to 2 vol% methacrylic acid, 0.3 to 3 g / L quaternized chitosan, 0.5 vol% to 1.6 vol% crosslinking agent, 0.5 to 1.5 g / L initiator, and the balance being water.

[0016] Further, the concentration of the drug monomer solution in step (3) is 0.01–10 g / L. Preferably, the concentration of the drug monomer solution is 0.5–10 g / L.

[0017] The solvents used to prepare the drug monomer solution include, but are not limited to, water and PBS buffer solution.

[0018] Furthermore, the acrylate monomer is hydroxyethyl methacrylate (HEMA) or glyceryl methacrylate.

[0019] Furthermore, the pharmaceutical monomer is any one or at least two of the following: epigallocatechin gallate (EGCG), epicatechin, epigallocatechin, chlorogenic acid, gallic acid, rhodioloside, and salvianolic acid B.

[0020] Furthermore, the crosslinking agent is ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, or dicyclopentenyl acrylate.

[0021] Furthermore, the initiator is 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylpropanone I2959 or 2-hydroxy-2-methyl-1-phenyl-1-propanone D1173.

[0022] Furthermore, in step (2), the irradiation wavelength of the ultraviolet light-irradiated free radical polymerization reaction is 300-400 nm, and the light intensity is 10-1500 mW / cm². 2 The polymerization time is 15-40 min; the demolding temperature is 70-80℃; after demolding, the hydrogel is washed in pure water for 3-7 days, and the pure water is changed daily.

[0023] Further, in step (3), the freeze-dried hydrogel is immersed in a drug monomer solution in a shaker in the dark at a temperature of 15-40℃ and a rotation speed of 100 rpm for 24-72 hours. After it has fully swollen and absorbed the drug, the corneal contact lens is taken out, and the drug sustained-release multifunctional hydrogel corneal contact lens is obtained.

[0024] Secondly, the present invention seeks protection for the fast photocrosslinked drug-releasing multifunctional hydrogel corneal contact lens prepared by the above method.

[0025] Compared with the prior art, the present invention has the following significant advantages:

[0026] 1. This invention provides a rapid method for preparing hydrogel corneal contact lenses by photocrosslinking. Compared with traditional methods, this method is not affected by temperature, uses one-step photocrosslinking molding, and is simple and easy to operate.

[0027] 2. The drug-releasing multifunctional hydrogel contact lens prepared by this invention can be combined with the modified biomacromolecule material quaternized chitosan, which improves the performance drawbacks of traditional contact lenses while enhancing the basic physicochemical properties of the contact lens.

[0028] 3. The drug-releasing multifunctional hydrogel corneal contact lens prepared by this invention crosslinks the active ingredients of traditional Chinese medicine in a physical manner. Compared with traditional antibiotics, it is safer and does not cause drug resistance. At the same time, the antibacterial components of traditional Chinese medicine can be continuously released in a sustained-release manner, which significantly improves the bioavailability of ophthalmic drugs and has broad application prospects for the treatment of future ophthalmic diseases.

[0029] 4. The drug-releasing multifunctional hydrogel corneal contact lens prepared by this invention can combine the advantages of each monomer component and has multiple therapeutic effects such as anti-inflammatory, antioxidant, and antibacterial properties, providing a brand-new solution for the clinical treatment of bacterial keratitis. Attached Figure Description

[0030] Figure 1 The infrared spectra of the hydrogel corneal contact lenses in Examples 1-5 are compared.

[0031] Figure 2 A comparison chart of the water content of hydrogel corneal contact lenses in Examples 1-5;

[0032] Figure 3 This is a comparison chart of the light transmittance of the hydrogel corneal contact lenses in Examples 1-5;

[0033] Figure 4 The mechanical strength comparison diagrams of the hydrogel corneal contact lenses in Examples 1-5 are shown.

[0034] Figure 5 This is a comparison chart of the release curves of the hydrogel corneal contact lenses in Examples 2-5;

[0035] Figure 6 This is a comparison chart of the antioxidant properties of the hydrogel corneal contact lenses in Examples 1-5;

[0036] Figure 7 Comparison of the antibacterial properties of hydrogel corneal contact lenses in Examples 1-5 against Staphylococcus aureus and Escherichia coli;

[0037] Figure 8 The preferred embodiment of the hydrogel corneal contact lens and the model group, and the comparison of rat eyeballs after treatment with the basic contact lens and positive drug for bacterial keratitis;

[0038] Figure 9 This is a comparison chart of the immunofluorescence effects of hydrogel contact lenses in the example group and other groups after treatment of bacterial keratitis.

[0039] Figure 10 This is a quantitative comparison of the average fluorescence intensity of the hydrogel contact lenses in the example group and the immunofluorescence after treatment of bacterial keratitis in each group.

[0040] Figure 11 Comparison of cytotoxicity of hydrogel corneal contact lenses in Examples 1-5;

[0041] Figure 12 Comparative images of the hemolytic effect of hydrogel corneal contact lenses in Examples 1-5;

[0042] Figure 13This is an illustration of the eye irritation effect of a hydrogel corneal contact lens as an example. Detailed Implementation

[0043] The technical solution of the present invention will be further explained and illustrated below through specific embodiments.

[0044] Example 1

[0045] (1) Add 5 mL of HEMA monomer, 100 μL of methacrylic acid and 5 mg of chitosan quaternary ammonium salt to 3 mL of water, and sonicate and stir for 5-15 min at 25℃-30℃ until completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and sonication for 5-15 min until the initiator is completely dissolved to form a reaction mixture.

[0046] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain the freeze-dried gel.

[0047] (3) Soak the freeze-dried gel in PBS solution and incubate in a constant temperature shaker (37℃, 100rpm) in the dark for 48h until the hydrogel is completely swollen to obtain a hydrogel corneal contact lens without Chinese medicine monomer loading.

[0048] Example 2

[0049] (1) Add 5 mL of HEMA monomer, 100 μL of methacrylic acid and 5 mg of chitosan quaternary ammonium salt to 3 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture.

[0050] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain the freeze-dried gel.

[0051] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 3 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0052] Example 3

[0053] (1) Add 5 mL of HEMA monomer, 100 μL of methacrylic acid and 5 mg of chitosan quaternary ammonium salt to 3 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture.

[0054] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0055] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 5 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0056] Example 4

[0057] (1) Add 5 mL of HEMA monomer, 100 μL of methacrylic acid and 5 mg of chitosan quaternary ammonium salt to 3 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture.

[0058] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0059] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 8 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0060] Example 5

[0061] (1) Add 5 mL of HEMA monomer, 100 μL of methacrylic acid and 5 mg of chitosan quaternary ammonium salt to 3 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture;

[0062] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0063] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 10 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0064] Example 6

[0065] (1) Add 6 mL of HEMA monomer, 80 μL of methacrylic acid and 10 mg of chitosan quaternary ammonium salt to 2.5 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 90 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture;

[0066] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0067] (3) Soak the freeze-dried gel in PBS solution and incubate in a constant temperature shaker (37℃, 100rpm) in the dark for 48h until the hydrogel is completely swollen to obtain a hydrogel corneal contact lens without Chinese medicine monomer loading.

[0068] Example 7

[0069] (1) Add 6 mL of HEMA monomer, 110 μL of methacrylic acid and 15 mg of chitosan quaternary ammonium salt to 2.5 mL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 60 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture;

[0070] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0071] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 3 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0072] Example 8

[0073] (1) Add 6 mL of HEMA monomer, 90 μL of methacrylic acid and 25 mg of chitosan quaternary ammonium salt to 3090 μL of water, and treat with ultrasound and stirring for 5-15 min at a temperature of 25℃-30℃ until the chitosan quaternary ammonium salt is completely dissolved; then add 80 μL of crosslinking agent ethylene glycol dimethacrylate and 10 mg of photoinitiator I2959 in sequence, and use stirring and ultrasound for 5-15 min until the initiator is completely dissolved to form a reaction mixture;

[0074] (2) Transfer the reaction mixture into a mold and expose it to ultraviolet light at a wavelength of 365 nm and an intensity of 1100 mW / cm². 2 Free radical polymerization was carried out at a reaction temperature of 25℃ for 20 minutes. After the reaction, the mold was immersed in hot water at 70℃, the hydrogel was demolded and washed in pure water for 3 days, with the pure water changed daily. The washed hydrogel was then freeze-dried to obtain a freeze-dried gel.

[0075] (3) The freeze-dried gel was soaked in an aqueous solution of epigallocatechin gallate (EGCG) with a concentration of 3 g / L, and then soaked and shaken in a constant temperature shaker (37℃, 100 rpm) in the dark for 48 h until the hydrogel was completely swollen to obtain a hydrogel corneal contact lens loaded with Chinese medicine monomer.

[0076] The following tests were conducted on the performance of the hydrogels from Examples 1-5.

[0077] I. Moisture content test:

[0078] The prepared hydrogel corneal contact lens was cut into identical circular pieces (15 mm in diameter). The cut lenses were then immersed in standard saline (phosphate buffered saline) water until they completely swelled and their weight no longer increased. The surface moisture was absorbed with filter paper, and the wet weight was recorded as Ws. The lenses were then dried in a 60°C oven for 48 hours. The dry weight Wd of the hydrogel lens was recorded when the dry weight no longer changed. The equilibrium water content EWC of the hydrogel was calculated using the following formula:

[0079]

[0080] II. Light transmittance test:

[0081] Using a UV-Vis spectrophotometer, the transmittance of swollen samples was measured in the wavelength range of 380–780 nm, with pure water as a control and wavelength intervals of 1 nm, to obtain the transmittance at different wavelengths.

[0082] III. Mechanical Strength Test:

[0083] Dumbbell-shaped hydrogel samples were prepared (test length 16 mm, test width 4 mm, tail width 8.5 mm, thickness 1 mm). Different groups of prepared hydrogel corneal contact lenses were immersed in physiological saline for 48 h (complete swelling). The surface moisture was absorbed with dust-free filter paper. The tensile strength of the swollen hydrogel samples was measured using an electronic universal testing machine at a stretching rate of 50 mm / min to test the tensile properties of the hydrogel.

[0084] IV. Drug loading and release kinetics:

[0085] The mass of the freeze-dried hydrogel was recorded as m1. The dried hydrogel was placed in a 50 mL centrifuge tube, and EGCG aqueous solution with volumes V0 = 5 mL and concentrations C0 of 3, 5, 8, and 10 g / L were added. The centrifuge tube was then placed in a constant temperature shaker (37℃, 100 rpm) and immersed in the dark with shaking until complete swelling. After complete swelling, the hydrogel was freeze-dried and the weight recorded as m2. The drug loading rates of different groups of contact lenses are calculated as follows:

[0086]

[0087] After soaking, the samples were wiped clean of drug solution with lint-free paper and placed in 50 mL centrifuge tubes. V1 = 6 mL of PBS solution (0.1 M, pH = 7.4) was added. At regular intervals, V2 = 3 mL of solution was taken from the centrifuge tubes, and 3 mL of fresh PBS solution was added. The absorbance (A) of the solution taken at each time point was measured at the characteristic wavelength (274 nm), and the concentration (C) was calculated using the standard curve of the drug solution. i The cumulative drug release from the hydrogel was calculated using the following formula:

[0088]

[0089] V. Antioxidant assay:

[0090] The DPPH method is used to determine the antioxidant capacity of hydrogels: Because the 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) radical has a single electron, it can accept an electron or a hydrogen ion, exhibiting maximum absorption at 517 nm, and its alcoholic solution turns purple. In the presence of antioxidants, due to an electron transfer mechanism, the unpaired electrons (nitrogen radicals) in DPPH are removed, causing the solution color to lighten. The degree of fading is quantitatively related to the degree of removal (i.e., the change in absorbance). Therefore, the antioxidant capacity of hydrogel contact lenses can be measured by their DPPH radical scavenging rate.

[0091] A 10 mg sample of hydrogel corneal contact lens was added to 30 mL of freshly prepared DPPH / ethanol solution (0.1 mM), and the mixture was incubated in the dark at 37°C and 100 rpm for 20 min. DPPH was evaluated using the following formula. + Clearance rate.

[0092]

[0093] Wherein, Ai, Aj, and Ac are the absorbance at 517 nm of the free radical solution mixed with the sample, the absorbance of the sample without DPPH solution, and the absorbance of the DPPH free radical solution without the sample, respectively. Each group of samples was tested 3 times, and the results were averaged.

[0094] The antioxidant capacity of the hydrogel was determined by the ABTS method: 0.2 mL of ABTS diammonium salt aqueous solution (7.4 mM) and 0.2 mL of K2S2O8 aqueous solution (2.6 mM) were mixed and reacted at room temperature in the dark for 12 h. Before use, the solution was diluted with anhydrous ethanol to achieve an absorbance of 0.70 ± 0.02 at 752 nm. Then, 10 mg of hydrogel corneal contact lens sample was added to 30 mL of freshly prepared ABTS solution. + The mixture was placed in a solution and kept in the dark at 37°C and 100 rpm for 40 minutes. ABTS + The calculation method for free radical scavenging rate is similar to that for DPPH· scavenging rate. Each sample group was tested three times, and the results were averaged.

[0095] VI. Antibacterial properties:

[0096] After sterilization, place the sample in a 12-well plate and take 1 mL of a 10⁻⁶ concentration. 6 CFU / mL Staphylococcus aureus (Escherichia coli) bacterial suspension was added to different hydrogel corneal contact lens samples, immersing the hydrogel sheets in the bacterial suspension. Simultaneously, 1 mL of bacterial suspension was added to each well of a hydrogel-free microplate as a control group, and 1 mL of culture medium was added to each well as a blank group. After inoculation, the wells were sealed and incubated for 3 hours at 37°C, 100 rpm, and 90% relative humidity in a constant-temperature shaker. Then, 200 μL of each group was transferred to a 96-well plate and its OD value was measured using a preheated microplate reader. Based on the measured OD value (approximately 0.1), 100 μL of each group was added to 10 mL of culture medium, mixed thoroughly, and then diluted using a 10-fold serial dilution method (1 mL added to 9 mL of culture medium). Take 100 μL of each of the different groups of diluted bacterial solutions and place them on solid culture dishes. Use a spreader to distribute the bacterial solutions evenly in the culture dishes. After sealing, incubate upside down in a 37℃ oven for 16-18 hours.

[0097] VII. Animal Experiments:

[0098] SD rats were selected to establish a bacterial keratitis (BK) model. The experimental animals were approved by the Ethics Committee of China Pharmaceutical University and the procedures were conducted in accordance with the "Guidelines for the Protection and Use of Laboratory Animals of China Pharmaceutical University". Under constant temperature and humidity conditions, the rats were allowed unrestricted food and water intake and were acclimatized for one week.

[0099] (1) Method for establishing the BK model

[0100] Before any intervention, the ocular surface and eyeballs of the rats were observed, recorded, and photographed. The collection time for these images was defined as day 0. The rats were then completely anesthetized by intraperitoneal injection of a freshly prepared 20% urethane solution (injection dose not exceeding 0.5 mL / 100 g). The left eye was selected as the target eye for modeling in all groups. Simultaneously, the surface anesthetic promecaine hydrochloride was instilled into one eye, and then the corneal epithelium of the rat was scraped away with a scalpel. The scraped area was approximately a circle centered on the pupil, with a diameter of about 2 mm. Afterwards, 50 μL of 1×10⁻⁶ promecaine hydrochloride solution was instilled into the scraped area. 8 After administering CFU / mL Staphylococcus aureus solution, the rats were kept with their eyes closed for 24 hours. The ocular surface infection status of the rats was observed using an electron vertical microscope, and photographs were taken and recorded as day 1. Simultaneously, corneal clarity and ocular surface inflammation were scored, and the severity of infection was assessed and recorded. The scoring criteria are shown in Table 1.

[0101] Table 1. Symptom grading and scoring criteria in a rat model of infectious keratitis.

[0102]

[0103]

[0104] (2) Experimental Grouping

[0105] Twenty rats with similar scores were randomly divided into four groups. The first group served as a control group, receiving no intervention. The second group received treatment with pHEMA hydrogel (basic contact lens group, conventional contact lens sample), wearing it for no more than 8 hours daily. After each treatment, the hydrogel was removed, ultrasonically cleaned with sterile water, and reused the following day. The third group received antibiotic treatment with levofloxacin eye drops (levofloxacin eye drops group), administered 1 drop three times daily. The fourth group received the same treatment as the pHEMA hydrogel group, using the hydrogel corneal contact lens from Example 2 (example group). Subsequently, the rats' ocular structure and the progression of corneal inflammation were observed every two days, and photographs were taken and scores recorded on days 3, 5, and 7. The scoring table is shown in Table 1.

[0106] VIII. Immunofluorescence Experiment:

[0107] On the seventh day after treatment, rats in each group were euthanized. Eyeballs were removed and stored in 20 times their volume of general-purpose fixative for over 24 hours to ensure complete fixation of the ocular tissue. Under a stereomicroscope, intact rat corneal tissue was completely removed from the eyeball, and excess conjunctiva, iris, and ciliary body tissue was trimmed. The corneal tissue was dehydrated overnight using an automated dehydrator. After embedding and fixation, paraffin-embedded corneal tissue sections were placed in a retrieval chamber filled with citrate antigen retrieval solution (pH 6.0), heated for 30 min, and then allowed to cool naturally to room temperature. The sections were washed three times with PBS for 5 min each time; then, an appropriate amount of 3% bovine serum albumin blocking solution was added, evenly covering the tissue, and incubated at room temperature for 30 min. The BSA blocking solution was discarded, and an appropriate amount of PBS-diluted primary antibody (IL-6, IL-1β, and TNF-α) working solution was added to the tissue, evenly covering it. The sections were then placed flat in a humidified chamber and incubated overnight at 4°C. The next day, the sections were removed from the refrigerator and allowed to thaw at room temperature for 30 min. Add an appropriate amount of PBS to the secondary antibody (Cy) that is diluted with the primary antibody and corresponds to its species. TM 3AffiniPure Donkey Anti-Mouse IgG(H+L) and Cy TM 3. Affini Pure Donkey Anti-Rabbit IgG (H+L) was incubated at room temperature for 1 hour. The slides were then immersed in autofluorescence quencher A solution and incubated at room temperature for 30 minutes. Freshly prepared DAPI working solution was then added, and the slides were incubated at room temperature in the dark for 10 minutes. Finally, a suitable amount of anti-quenching mounting adhesive was applied to the tissue, and a coverslip was placed on top. The slides were then observed under a scanner. The mean fluorescence intensity of each inflammatory factor was semi-quantitatively analyzed using ImageJ software (n=3).

[0108] IX. In vitro cytotoxicity experiments:

[0109] Different groups of hydrogel contact lenses, after swelling, were washed three times with sterile PBS and then sterilized under UV light for 4 hours (radiation wavelength 253 nm). Samples and complete DMEM (Dulbecco's modified eagle medium) were added to sterile 6-well plates at a ratio of 20 mg / mL (sample / DMEM), and the plates were placed in a sterile environment at 37°C. After 24 hours of sample extraction, the extract was collected and mixed with 10% (v / v) FBS serum and 1% (v / v) penicillin antibody to prepare DMEM complete medium. After cell counting, the medium was diluted to 5 × 10⁶ cells / mL. 4 Add 100 μL of cell suspension (approximately 5 × 10⁶ cells / mL) to each well of a 96-well plate. 3Cells were collected and then returned to a cell culture incubator (37℃, 5% CO2, 90% relative humidity) for 24 hours. When the cells reached a density of 80-90%, they were washed with sterile PBS and 100 μL of the prepared DMEM complete medium was added to continue culturing. DMEM without extract was used as a negative control. After 24 hours of culture, the medium and hydrogel were discarded, and the cells were washed again with sterile PBS. 110 μL of CCK-8 solution diluted 10:1 with the medium was added, and the cells were returned to the incubator in the dark for 1-1.5 hours. Finally, the absorbance A was measured at a detection wavelength of 450 nm, and the cell viability was calculated using the following formula.

[0110]

[0111] Where A is the absorbance of cells cultured in the sample culture medium; A blank is the absorbance of complete culture medium solution with 10% CCK-8 added; and A control is the absorbance of the negative control group.

[0112] 10. Hemolysis test:

[0113] 40 μL of red blood cells were transferred to a 15 mL centrifuge tube, and 2 mL of deionized water was added to prepare a 2% (v / v) red blood cell suspension as a positive control. 1 mL of the previously diluted 4% red blood cell suspension was transferred to a 15 mL centrifuge tube, and 1 mL of PBS buffer was added to dilute it to a 2% red blood cell suspension as a negative control. Hydrogel contact lenses from different groups were washed, cut to the same size (r = 1.5 cm), and placed in 15 mL centrifuge tubes. 1 mL of PBS was added, and the tubes were shaken well. Then, 1 mL of 4% red blood cell suspension was added to each sample, and each sample was diluted to a 2% red blood cell suspension. The control and sample groups were incubated in a 37°C water bath for 3 hours at 4000 rpm. -1 Centrifuge for 5 min, collect the supernatant, and measure the absorbance of different groups at 540 nm using a UV spectrophotometer. Calculate the hemolysis rate using the following formula: DS represents the absorbance of the experimental group, Dn represents the absorbance of the negative control, and Dw represents the absorbance of the positive control.

[0114]

[0115] XI. Eye Stimulation Test:

[0116] Using a circular punch, the contact lenses from the example group, after swelling in the drug solution, were cut to a size similar to the rat's eyeball (R = 0.8 mm), sterilized with ultraviolet light, and then worn on the healthy eyes of rats. They were removed after 8 hours of wear each day, and the condition of the rat's eyeball was immediately recorded. This process was repeated for three consecutive days, and the results were recorded as observations of eye stimulation.

[0117] Experimental results:

[0118] Table 2. Drug loading results for each case group

[0119]

[0120] As shown in Table 2, the drug loading rate of the contact lenses prepared in each example gradually increased with the increase of EGCG drug concentration. Different drug concentrations may have resulted in different crosslinking densities, leading to differences in drug loading.

[0121] like Figure 1 As shown, the hydrogel contact mirrors of Examples 1-5 form a stable physical cross-linked network through hydrogen bonds and cation-π interactions. Specifically, EGCG is located at 3349 cm⁻¹ in the spectrum. -1 The region exhibits characteristic peaks of -OH stretching vibration, and from 1689 cm⁻¹... -1 and 1613cm -1 The characteristic peak of the C=O stretching vibration indicates that EGCG is rich in hydroxyl groups. With the addition of obfEGCG, the peak shapes of Examples 2, 3, 4, and 5 broaden, showing a blue shift in peak value, up to 3394 cm⁻¹. -1 Compared to Example 1, the peak signal intensity was enhanced, indicating the formation of hydrogen bonds in the hydrogel network; meanwhile, the peak signal intensity of Example 1 was significantly higher at 1610 cm⁻¹. -1 and 740cm -1 The presence of characteristic peaks at the left and right positions indicates the presence of benzene ring C=C and benzene ring CH; furthermore, in each set of examples, the -C=O vibration of EGCG decreased from 1689 cm⁻¹. -1 The shift to higher wavenumbers can be attributed to the interaction between -C=O and the hydrogen donor, further confirming the loading of the EGCG monomer. In the various hydrogel spectra, 1476 cm⁻¹... -1 The shift of the -CH bond of the quaternized methyl group in the QCS to a lower wavenumber indicates the presence of hydrogen bonding and cation-π interaction, thus proving the loading of the QCS.

[0122] like Figure 2 As shown, the water content of the hydrogel contact lenses in Examples 1-5 is greater than 40%, which meets the requirements of the national standard (GB11417), proving that the hydrogel contact lenses in Examples 1-5 have good water content.

[0123] like Figure 3 As shown, the hydrogels of Examples 1-5 all have a transmittance of more than 90% in the visible light wavelength range, proving that the hydrogel contact lenses in Examples 1-5 have excellent light transmittance.

[0124] like Figure 4As shown, in the hydrogel contact lenses of Examples 1-5, after physical cross-linking with traditional Chinese medicine monomers, their mechanical properties are significantly improved, exhibiting good tensile strength and elongation at break, thus ensuring the durability and reliability of the contact lenses.

[0125] like Figure 5 As shown, the hydrogel contact lenses of Examples 2-5 have a sustained drug release effect and can continuously release drugs within 60 hours.

[0126] like Figure 6 As shown, the hydrogel contact lenses of Examples 1-5 were tested and tested using DPPH· and ABTS·. + Experiments evaluated the free radical scavenging ability of the hydrogel, confirming the antioxidant capacity of the drug-loaded hydrogel contact lens. The antioxidant effect of the contact lens gradually increased with increasing drug concentration.

[0127] like Figure 7 As shown, the hydrogel contact lenses of Examples 1-5 demonstrated good antibacterial activity against Staphylococcus aureus and Escherichia coli using the plate count method.

[0128] like Figure 8 As shown, in vivo pharmacodynamic studies were conducted on the hydrogel contact lens of preferred embodiment 3. Compared with treatment with the positive drug levofloxacin eye drops, the corneal tissue treated with the contact lens of embodiment 3 recovered rapidly, demonstrating a superior therapeutic effect.

[0129] like Figure 9 As shown, after treating animal models with the hydrogel contact lenses in Example 3, immunofluorescence assays were performed on the corneal tissue. IL-6, IL-1β, and TNF-α are representative inflammatory factors associated with infection-related inflammatory responses. Compared to the significant fluorescent expression of inflammatory factors in other groups, the inflammatory factors in Example 3 were almost not expressed, indicating that the hydrogel contact lenses have a significant anti-inflammatory effect in treating bacterial keratitis.

[0130] like Figure 10 As shown, a semi-quantitative analysis of the relative average fluorescence intensity of IL-6, IL-1β and TNF-α in each group was further performed in immunofluorescence. The inflammatory fluorescence intensity of the 3rd group after treatment was significantly different from that of the other groups, further demonstrating its excellent anti-inflammatory effect.

[0131] like Figure 11 As shown, the hydrogel contact lenses of Examples 1-5 all showed a cell survival rate of over 80% in cytotoxicity tests, demonstrating that the material has good cell compatibility.

[0132] like Figure 12As shown, the hydrogel contact lenses of Examples 1-5 all showed a hemolysis rate of less than 5% in the hemolysis test, proving that the material has good blood compatibility.

[0133] like Figure 13 The images shown are from Example 2, illustrating the ocular irritation effects of the hydrogel contact lenses. During continuous observation, there were no significant differences in the rats' eyes before and after wearing the contact lenses. The eye structure remained intact, the corneal parenchyma was clear and transparent without cloudiness, and the conjunctival vessels were clearly visible without significant congestion. This indicates that the contact lenses did not irritate the rats' eyes in the short term, did not cause any acute adverse ocular reactions, and possessed good biocompatibility.

[0134] The experimental results above demonstrate that the drug-releasing multifunctional hydrogel corneal contact lenses of the various embodiments of the present invention possess ideal water content, excellent light transmittance, and superior mechanical strength, enabling sustained drug release. They exhibit significant bioactivity in antioxidation, antibacterial activity, and anti-inflammation, particularly in the treatment of bacterial keratitis, where they show superior efficacy compared to levofloxacin eye drops. Furthermore, the biocompatibility of the material has been consistently confirmed in in vitro and in vivo studies, demonstrating good biocompatibility and safety, making it a promising novel ophthalmic drug delivery system.

[0135] The above description is merely a detailed explanation of preferred embodiments and principles of the present invention. For those skilled in the art, there may be changes in specific implementation methods based on the ideas provided by the present invention, and these changes should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a fast photo-crosslinking drug-releasing multifunctional hydrogel contact lens, characterized in that, The preparation method comprises the following steps: (1) proportionally adding quaternary ammonium chitosan, acrylate monomer and methacrylic acid into water, and using ultrasonic and stirring methods to completely dissolve the quaternary ammonium chitosan; then sequentially adding a crosslinking agent and an initiator, and using ultrasonic and stirring methods to completely dissolve the initiator to form a reaction mixture; (2) transferring the reaction mixture into a mold, performing ultraviolet light irradiation free radical polymerization, after the reaction is completed, demolding to take out the hydrogel and washing, and performing freeze-drying treatment on the washed hydrogel to obtain a freeze-dried hydrogel; (3) immersing the freeze-dried hydrogel in a drug monomer solution until the hydrogel is completely swelled to obtain a drug sustained-release multifunctional hydrogel contact lens; The acrylate monomer is hydroxyethyl methacrylate or glycerol methacrylate; The drug monomer is any one or at least two of epigallocatechin gallate, epicatechin, epigallocatechin, chlorogenic acid, gallic acid, rhodioloside and salvianolic acid B.

2. The method of claim 1, wherein, In step (1), the content of each component in the reaction mixture is as follows: 30 vol% to 80 vol% of the acrylate monomer, 0.6 vol% to 2 vol% of the methacrylic acid, 0.001 to 3 g / L of the quaternary ammonium chitosan, 0.5 vol% to 1.6 vol% of the crosslinking agent, 0.01 to 1.5 g / L of the initiator, and the balance being water.

3. The method of claim 1, wherein, In step (3), the concentration of the drug monomer solution is 0.01 to 10 g / L.

4. The method according to claim 1 or 2, characterized in that, The crosslinking agent is ethylene glycol dimethacrylate, triethylene glycol dimethacrylate, tetraethylene glycol diacrylate, polyethylene glycol dimethacrylate or dicyclopentenyl acrylate.

5. The method according to claim 1 or 2, characterized in that, The initiator is 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone I2959 or 2-hydroxy-2-methyl-1-phenyl-1-propanone D1173.

6. The method of claim 1, wherein, In step (2), the wavelength of the ultraviolet light for the radical polymerization is 300-400 nm, the light intensity is 10-1500 mW / cm 2 , and the polymerization time is 15-40 min; the temperature for the demolding is 70-80℃, and the hydrogel is washed in pure water for 3-7d after the demolding.

7. The method of claim 1, wherein, In step (3), the freeze-dried hydrogel is immersed in the drug monomer solution and shaken in a shaking bed in the dark, the immersion temperature is 15-40℃, the rotation speed is 100 rpm, and the immersion time is 24-72 h.

8. The fast photo-crosslinking drug sustained-release multifunctional hydrogel contact lens prepared by the method in any one of claims 1-7.

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