Hydrogel for resisting scarring reaction after glaucoma filtration operation as well as preparation method and application of hydrogel

Abstract

The invention relates to hydrogel for resisting a scarring reaction after a glaucoma filtration operation as well as a preparation method and application of the hydrogel. The hydrogel comprises the following components: 2-hydroxyethyl methylacrylate and 2-((2-hydroxy-3-(methylacryloyloxy) propyl) dimethylamine) acetate. The hydrogel has good optical performance, mechanical performance and biological antifouling performance. When the hydrogel is applied to rabbit eye glaucoma filtration operation, the rabbit intraocular pressure can be effectively reduced, filtration vesicles can be maintained, and meanwhile, the cicatrization reaction of rabbit sclera flaps is remarkably relieved. The hydrogel can also be used as a drug carrier platform to load anti-cicatrization drugs, hormone drugs and the like, and a new strategy is provided for clinical application of anti-cicatrization after glaucoma filtration.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to a novel hydrogel for resisting scarring reaction after glaucoma filtration surgery (GFS) and a preparation method and application thereof. Background Art

[0002] Glaucoma surgery is generally considered to be an effective treatment for patients whose intraocular pressure cannot be effectively controlled by anti-glaucoma drugs or laser treatment. Among them, GFS is one of the common surgical methods for glaucoma. Its success depends on the efficiency of external drainage of aqueous humor, and its efficacy depends on incomplete healing of surgical wounds. However, the damage caused by GFS stimulates the proliferation and differentiation of fibroblasts and the excessive deposition of extracellular matrix (ECM) in the filtration area. Scar formation in the surgical area is an important factor limiting the success rate of GFS. Uncontrolled intraocular pressure after GFS may lead to further deterioration of glaucomatous optic disc depression and subsequent visual field loss. In clinical practice, antimetabolites such as mitomycin C (MMC) and 5-fluorouracil (5-Fu) are often used to inhibit scar formation. However, the use of these drugs must be strictly controlled within an appropriate dosage range. Excessive or improper use may bring a series of complications, accompanied by the risk of many complications, such as persistent hypotony, corneal toxicity, filtration bleb leakage and endophthalmitis, which may cause permanent visual impairment in patients. Therefore, developing new anti-scarring strategies with greater anti-fibrotic effects and lower cytotoxicity is crucial to improving the safety and effectiveness of GFS surgery.

[0003] 2-((2-Hydroxy-3-(methacryloyloxy)propyl)dimethylammonium) acetate (CBOH) is known for its excellent bio-antifouling properties. As a zwitterion, CBOH can interact with water molecules through the ion solvation effect to form a hydration layer on the surface of the material, giving it superhydrophilicity, thereby effectively reducing the adsorption of nonspecific proteins and cell adhesion, opening up new possibilities for anti-scarring treatment. However, PCBOH lacks toughness. If its mechanical properties can be improved through modification, it will be able to better meet the requirements for material suturing in GFS surgery, thereby enhancing its applicability in clinical applications.

[0004] 2-Hydroxyethyl methacrylate (HEMA) is valued for its good mechanical properties and biocompatibility. It is similar to the side chain structure of CBOH monomer and is easier to combine with CBOH. The present invention combines HEMA monomer with CBOH monomer, which not only effectively solves the problem of insufficient lubricity and foreign body reaction that may be caused by long-term implantation, but also improves the flexibility of the hydrogel and meets the demand for material suturing in GFS surgery. Therefore, the research and development of P (HEMA-CBOH) hydrogel provides an important idea for the innovation of new anti-scarring treatment methods in GFS surgery. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of scarring after GFS surgery and to provide a novel hydrogel for alleviating scarring reaction after glaucoma filtration surgery and a preparation method and application thereof.

[0006] The present invention successfully prepares P (HEMA-CBOH) zwitterionic hydrogel. P (HEMA-CBOH) hydrogel contains HEMA monomer and CBOH monomer. The polymer PCBOH of CBOH has good biofouling resistance, and its superhydrophilicity can effectively reduce the attachment and adsorption of fibroblasts, and reduce the inflammatory response and fibrous capsule formation that may occur after GFS surgery. The polymer of HEMA (PHEMA) has good mechanical properties and biocompatibility, which can meet the requirements of material suturing in GFS surgery, and ensure the convenience of surgical operation and the stability of postoperative recovery. The research results of the present invention show that P (HEMA-CBOH) hydrogel has good optical properties, mechanical properties and biofouling resistance, can effectively maintain the postoperative effect of GFS and reduce the scarring reaction of rabbit eyes after GFS surgery, and this method provides a new strategy for promoting the clinical application of anti-scarring after GFS surgery. In addition, the hydrogel can be used as a drug carrier platform to load anti-scarring, hormones and other drugs, laying a solid foundation for further clinical application and promotion.

[0007] The technical solution of the present invention is: A method for preparing a hydrogel for resisting scarring reaction after glaucoma filtration surgery comprises the following steps: 1) Heat and dissolve the CBOH monomer in the HEMA monomer solution to form a uniform medium solution; 2) adding 2-hydroxy-2-methyl-1-phenyl-1-propanone (photoinitiator 1173) and chemical crosslinking agent triethylene glycol dimethacrylate (TEGDMA) to the solution obtained in step 1), and mixing at room temperature to obtain a HEMA-CBOH solution; 3) The HEMA-CBOH solution obtained in step 2) was mixed thoroughly and transferred into a mold, and irradiated under 365 nm UV light (predetermined light intensity) for 1 hour to obtain P(HEMA-CBOH) hydrogel.

[0008] 4) The P(HEMA-CBOH) hydrogel obtained in step 3) was immersed in 75% ethanol overnight, and dialyzed in PBS for at least 4 days to remove residual small molecules to obtain a sterile hydrogel.

[0009] In the present invention, the mass ratio of the CBOH monomer is 0.1-15 wt% (relative to the total mass), more preferably 5-13 wt% (relative to the total mass), and most preferably 12 wt% (relative to the total mass).

[0010] In the present invention, the heating temperature in step 1) is 80-100 degrees, and the heating time is 2-5 minutes, and the most preferred heating time is 80 degrees for 3 minutes.

[0011] In the present invention, the mass ratio of the photoinitiator 1173 is 0.1-5 wt % relative to the total mass of the solution, more preferably 0.5-3 wt %, and most preferably 1 wt %. The mass ratio of the chemical crosslinking agent TEGDMA is 0.1-1 wt % relative to the total mass, more preferably 0.2-0.6 wt %, and most preferably 0.5 wt %.

[0012] In the present invention, the thickness of the P(HEMA-CBOH) hydrogel is 100-1000 um, more preferably 100-500 um, and most preferably 100 um.

[0013] The present invention provides a novel hydrogel for resisting scarring reaction after glaucoma filtration surgery, which can be loaded with anti-scarring drugs, hormones and the like.

[0014] The raw materials used in the present invention can be conventional commercially available products, and the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0015] The technical effects of the present invention are: CBOH is known for its excellent bio-antifouling properties. As a zwitterion, CBOH can interact with water molecules through the ion solvation effect to form a hydration layer on the surface of the material, giving it super-hydrophilicity, thereby effectively reducing the adsorption of nonspecific proteins and the adhesion of cells, opening up new possibilities for anti-scarring treatment. However, PCBOH is not flexible enough, and its mechanical properties need to be improved to meet the requirements for material sutureability in GFS. In order to overcome this limitation, the present invention introduces HEMA, which has a side chain structure similar to that of CBOH monomer. HEMA is valued for its good mechanical properties and biocompatibility, but the use of HEMA hydrogel alone still has some defects, such as low water content, severe foreign body sensation caused by high modulus, insufficient lubricity, and severe foreign body reaction that may be caused by long-term implantation. The present invention combines HEMA monomer with CBOH monomer to successfully synthesize a new type of zwitterionic hydrogel P (HEMA-CBOH). This hydrogel not only inherits the excellent mechanical properties of PHEMA, but also integrates the bio-antifouling properties of PCBOH, effectively solving the problems of insufficient lubricity and foreign body reaction that may be caused by long-term implantation. At the same time, it also improves the flexibility of the hydrogel, meeting the need for material suturing in GFS surgery. Therefore, the research and development of P (HEMA-CBOH) hydrogel provides an important idea for the innovation of new anti-scarring treatment methods in GFS surgery.

[0016] The mass ratio of CBOH monomer in P (HEMA-CBOH) hydrogel directly affects the water content of the hydrogel. In the present invention, the mass ratio of CBOH monomer is 0.1-15 wt% (relative to the total mass), more preferably 5-13 wt% (relative to the total mass), and most preferably 12 wt% (relative to the total mass). With the increase of zwitterionic CBOH content, the water content of the gel increases, resulting in a decrease in modulus, effectively reducing the foreign body sensation that may occur after surgery and improving comfort. 1173 and TEGDMA are used as photoinitiators and crosslinkers, and their mass ratios affect the mechanical properties and chemical stability of the hydrogel. In the present invention, the mass ratio of the photoinitiator 1173 is 0.1-5 wt% relative to the total mass of the solution, more preferably 0.5-3 wt%, and most preferably 1 wt%. The mass ratio of the chemical crosslinker TEGDMA is 0.1-1 wt% relative to the total mass, more preferably 0.2-0.6 wt%, and most preferably 0.5 wt%.

[0017] The present invention uses rabbit eyes to perform GFS. P (HEMA-CBOH) hydrogel can effectively reduce intraocular pressure, maintain the stability of the filtration bleb, significantly reduce foreign body reactions such as inflammation and fibrous capsule formation, reduce scleral collagen deposition, alleviate GFS surgical scar formation, and improve the success rate of GFS. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the synthesis of P(HEMA-CBOH) hydrogel.

[0019] Figure 2 The transmittance (A) and equilibrium water content (B) of P(HEMA-CBOH) hydrogel in the wavelength range of 400-800 nm.

[0020] Figure 3 Tensile strength and modulus of P(HEMA-CBOH) hydrogel.

[0021] Figure 4 This is the crack insensitivity test of P(HEMA-CBOH) hydrogel.

[0022] Figure 5 Protein adsorption characterization data of P(HEMA-CBOH) hydrogel (A), relative cell viability (B), and anti-cell adhesion ability (C).

[0023] Figure 6 Schematic diagram of P(HEMA-CBOH) hydrogel application in eyes.

[0024] Figure 7 This is the change of intraocular pressure before GFS operation and 3 days, 7 days, 14 days and 28 days after operation in rabbits.

[0025] Figure 8 The rabbits were examined by UBM 7, 14 and 28 days after GFS surgery.

[0026] Fig. 9 HE staining of rabbit eyeballs 7, 14 and 28 days after GFS surgery.

[0027] Fig.10 Masson staining of rabbit eyeballs 7, 14 and 28 days after GFS surgery.

[0028] Fig.11 The expression of a-SMA in rabbits 7, 14 and 28 days after GFS surgery. DETAILED DESCRIPTION

[0029] Example 1: Preparation of P(HEMA-CBOH) hydrogel Reagents and their sources: 2-Hydroxyethyl methacrylate (HEMA) and 2-hydroxy-2-methylpropenoate (IRGACURE 1173, 97%) were purchased from Tianjin Xiens Biochemical Technology Co., Ltd., and tetraethylene glycol diacrylate (TEGDMA) was purchased from Shanghai Myril Co., Ltd.

[0030] Preparation steps: At 80 °C, different masses of CBOH monomers were mixed and stirred with HEMA monomer solutions for 3 min to form uniform transparent solutions with CBOH monomer mass ratios of 0, 3, 6 or 12 wt% (relative to the total mass). Photoinitiator 1173 (relative to 1wt% of the total mass) and chemical crosslinker TEGDMA (relative to 0.5 wt% of the total mass) were added to the above solution and stirred evenly at room temperature. PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6, and P(HEMA-CBOH)-12 hydrogels were formed by irradiation with 365 nm UV light (predetermined light intensity) for 1 hour. The obtained hydrogel was soaked in 75% ethanol overnight, dialyzed in PBS to remove residual small molecules for at least 4 days, and then soaked in sterile PBS to obtain sterile hydrogels for use.

[0031] The preparation process of P(HEMA-CBOH) hydrogel is as follows Figure 1 shown.

[0032] Example 2: Preparation of P(HEMA-CBOH) hydrogel Different from Example 1, at 100 ° C, different masses of CBOH monomers were mixed and stirred with HEMA monomer solutions for 2 minutes to form a uniform transparent solution with a CBOH monomer mass ratio of 5 and 15 wt% (relative to the total mass). Photoinitiator 1173 (3 wt% relative to the total mass) and chemical crosslinker TEGDMA (0.1 wt% relative to the total mass) were added to the above solution and stirred evenly at room temperature. Irradiate under 365 nm UV light (predetermined light intensity) for 1 hour to form a hydrogel. The obtained P(HEMA-CBOH)-5 and P(HEMA-CBOH)-10 hydrogels were soaked in 75% ethanol overnight, dialyzed in PBS to remove residual small molecules for at least 4 days, and then soaked in sterile PBS to obtain sterile hydrogels for standby use.

[0033] Example 3: Preparation of P(HEMA-CBOH) hydrogel Different from Examples 1 and 2, different masses of CBOH monomers were mixed and stirred with HEMA monomer solutions at 90 °C for 5 min to form a uniform transparent solution with a CBOH monomer mass ratio of 0.1, 1 or 10 wt% (relative to the total mass). Photoinitiator 1173 (0.1wt% relative to the total mass) and chemical crosslinker TEGDMA (1wt% relative to the total mass) were added to the above solution and stirred evenly at room temperature. Irradiate under 365 nm UV light (predetermined light intensity) for 1 hour to form a hydrogel. The obtained P(HEMA-CBOH)-0.1, P(HEMA-CBOH)-1, and P(HEMA-CBOH)-10 hydrogels were soaked in 75% ethanol overnight, dialyzed in PBS to remove residual small molecules for at least 4 days, and then soaked in sterile PBS to obtain sterile hydrogels for standby use.

[0034] Example 4: Evaluation of Optical Properties and Measurement of Equilibrium Water Content of P(HEMA-CBOH) Hydrogel Test method: UV-Vis spectrophotometry and gravimetric method Test tool: UV-visible spectrophotometer (GENESYS 180, ThermoFisher Scientific) Experimental steps: Evaluation of optical properties: At room temperature, PBS was used as a blank and the transmittance of the hydrogels was measured in the wavelength range of 400-800 nm using a UV-visible spectrophotometer. PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6 and P(HEMA-CBOH)-12 hydrogels were cut into long strips and their transmittance was measured in a UV-visible spectrophotometer quartz dish.

[0035] Equilibrium water content measurement: The equilibrium water content (EWCs) of the hydrogel was determined by gravimetric method. The prepared hydrogel was placed in PBS for 4 days to reach swelling equilibrium, and then the hydrogel was taken out and weighed to obtain m wet , and then placed it in a freeze dryer to remove moisture and weigh it to get m dry Three parallel samples were prepared for each scale sample.

[0036] The EWCs of the hydrogels are:

[0037] like Figure 2 As shown in the figure, the prepared hydrogel has excellent optical properties. In addition, its high equilibrium water content helps to reduce the modulus, thereby significantly reducing the foreign body sensation during use and improving comfort.

[0038] Example 5: Tensile strength and modulus of P(HEMA-CBOH) hydrogel Test method: Uniaxial tensile test Test tool: Electronic universal testing machine (Legend 2344, Instron, USA) Experimental steps: Tensile test: PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6 and P(HEMA-CBOH)-12 hydrogels were prepared according to ASTM standards in dumbbell-shaped samples with a size of 35 × 2.1 × 1 mm. 3 All tests were performed at a constant rate of 50 mm / min. Young's modulus was calculated from the initial slope of the stress-strain curve in the strain range of 3% to 8%, and toughness was defined as the area under the stress-strain curve.

[0039] Too high tensile strength and modulus can lead to severe foreign body sensation after implantation in the eye, and the hydrogel used in GFS needs to be as soft as possible to meet suturing requirements. Figure 3 As shown, the performance indicators of each group of hydrogels are relatively low, which can effectively reduce the foreign body sensation after implantation. And with the increase of zwitterionic components, the flexibility of the hydrogel is more conducive to suturing.

[0040] Example 6: Crack insensitivity test of P(HEMA-CBOH) hydrogel Test method: Crack notch tensile cycle test Test tool: Electronic universal testing machine (Legend 2344, Instron, USA) Experimental steps: like Figure 4 As shown in A, the present invention evaluates the crack insensitivity of each group of hydrogels by crack notch tensile cycle test. In the loading-unloading tensile test, the notched specimens of P(HEMA-CBOH)-3, P(HEMA-CBOH)-6, and P(HEMA-CBOH)-12 hydrogels are first stretched to a predetermined strain (10%) at a tensile rate of 50 mm / min, and then immediately restored to the initial state at the same rate, wherein the notch length of the notched specimen is 15% of the specimen width, and its stability is observed.

[0041] like Figure 4 As shown in B, after 10 cycles of testing, the notched hydrogel showed excellent stability without any further crack expansion, which fully proved that the hydrogel was stable under tensile strain and could be sutured during surgery, making it suitable for glaucoma filtration surgery.

[0042] Example 7: Evaluation of biological properties of P(HEMA-CBOH) hydrogel Test method: BCA protein assay kit (Boster Biotechnology), CCK8 method (Dojindo, Japan) Testing tools: Tecan Spark multifunctional microplate reader, inverted fluorescence microscope (Eclipse Ti-SR, Nikon, Japan) Experimental steps: 1. Measurement of nonspecific protein adsorption: PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6 and P(HEMA-CBOH)-12 hydrogels in a state of swelling equilibrium were immersed in 1 mg / mL BSA, cultured at 37 °C for 1 d, and then rinsed with saline three times to remove the loosely adsorbed proteins on the surface of the hydrogels. Then, the proteins absorbed in the hydrogels were ultrasonicated in a 1% sodium dodecyl sulfate (SDS) aqueous solution for 10 minutes, determined using a BCA protein kit, detected using an enzyme reader, and the average value was recorded.

[0043] 2. Cell viability assay: Mouse fibroblasts (L929) were used to evaluate cell compatibility using the CCK-8 method. L929 cells were seeded in a 96-well plate at a density of 1×10 4 Cells / well were inoculated at 37 °C and 5% CO2 for 24 h. The culture medium was removed and PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6 and P(HEMA-CBOH)-12 hydrogel extracts containing culture medium were added, and untreated cells were used as controls. After incubation for 24 h, the cells were measured with CCK-8 kit, detected with a microplate reader, and the data were recorded.

[0044] 3. Determination of anti-cell adhesion ability: PHEMA, P(HEMA-CBOH)-3, P(HEMA-CBOH)-6 and P(HEMA-CBOH)-12 hydrogels were placed in a 48-well plate and incubated at 37°C for 1 h. L929 cells were cultured at 1×10 5 The cells were seeded into each well at a density of 10 cells / well and incubated at 37°C in 5% CO2 for 48 h. Then, the cells were gently rinsed with sterile PBS to remove the suspended cells. The L929 cells adhered to the hydrogel surface were stained with calcein AM and observed under an inverted fluorescence microscope.

[0045] In order to determine the antifouling ability of P(HEMA-CBOH)-3, P(HEMA-CBOH)-6, and P(HEMA-CBOH)-12 hydrogels, the present invention evaluated their nonspecific protein adsorption properties. Figure 5As shown in A, compared with pHEMA hydrogel, the resistance of each group of P(HEMA-CBOH) hydrogels to nonspecific protein adsorption was significantly improved.

[0046] like Figure 5 B shows that the cell viability of each group exceeded 98%, indicating that P(HEMA-CBOH) hydrogel has excellent biocompatibility.

[0047] like Figure 5 As shown in Figure C, after 48 hours of cell incubation, a large number of L929 cells were observed to adhere to the surface of the cell culture plate (TCPS). Similarly, there were a small number of L929 cells on the surface of the hydrophilic PHEMA hydrogel, while there was almost no attachment on the hydrogels of each group of P(HEMA-CBOH), indicating that it has excellent anti-cell adhesion ability.

[0048] Example 8: GFS surgery in rabbit eyes Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0049] Experimental equipment: Ophthalmic surgical microscope (YZ20P5, Suzhou Liuliu) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0050] 2. Trabecular group surgical method: Rabbits were anesthetized with thiazine hydrochloride (about 0.5 mL). The eyeball was exposed with a lid speculum and disinfected with iodine. Oxybuprocaine hydrochloride was used for local anesthesia of the ocular surface. The conjunctiva and subconjunctival tissue were carefully separated. Then, a 3*4 mm deep and two-thirds of the scleral thickness was made. 2 The rectangular scleral flap was soaked with 0.2 mg / mL MMC, covered with a sponge for 2 minutes and rinsed for 30 seconds, followed by trabeculectomy, and then the scleral flap and conjunctiva were intermittently sutured with 10-0 nylon sutures. TobraDex ointment and levofloxacin were used to reduce inflammation after surgery.

[0051] 3. Surgical method of hydrogel group: Rabbits were anesthetized with thiazine hydrochloride (about 0.5 mL). The eyeball was exposed with a lid speculum and disinfected with iodine. Oxybuprocaine hydrochloride was used for local anesthesia of the ocular surface. The conjunctiva and subconjunctival tissue were carefully separated. Then, a 3*4 mm deep and two-thirds thick sclera was made. 2 A rectangular scleral flap was created. The scleral flap was soaked with 0.2 mg / mL MMC, covered with a sponge for 2 minutes and rinsed for 30 seconds. Trabeculectomy was then performed, and the hydrogel was placed under the scleral flap. The scleral flap and conjunctiva were intermittently sutured with 10-0 nylon sutures. TobraDex ointment and levofloxacin were used to reduce inflammation after surgery.

[0052] Schematic diagram of the surgical procedure for the hydrogel group Figure 6 shown.

[0053] Example 9: GFS postoperative intraocular pressure measurement Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0054] Experimental equipment: Tonometer (TonoLab, ICare) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0055] 2. Before and after surgery, the IOP of the rabbits was measured by using a tonometer through the center of the cornea. To ensure accuracy, the measurement time was 2 pm. The intraocular pressure values ​​were measured before surgery and 3, 7, 14 and 28 days after surgery.

[0056] like Figure 7 As shown in the figure, 3 days after surgery, the intraocular pressure of the trabecular group and the material group decreased compared with the normal control group. Subsequently, 7 days after surgery, the antihypertensive effect of the hydrogel group was more obvious than that of the trabecular group. 28 days after surgery, the intraocular pressure of the trabecular group returned to normal levels, while the hydrogel group was able to continue to maintain the effect of lowering intraocular pressure, indicating that P(HEMA-CBOH) hydrogel can effectively reduce IOP.

[0057] Example 10: UBM measurement after GFS surgery Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0058] Experimental equipment: Ultrasonic biomicroscope (UBM) (MD-300L, MEDA Co., Ltd, Tianjin, China) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0059] 2. The rabbits were first anesthetized, and then the eyes were topically anesthetized with oxybuprocaine hydrochloride, and the bleb site was carefully exposed for clear imaging. The bleb was examined using an ultrasound biomicroscope and a covered sterile probe (QUANTEL MEDICAL, Clermont-Frand, France).

[0060] The filtration bleb is a structure formed during the GFS process and is one of the key indicators of surgical success. Figure 8As shown in the figure, the red arrow marks the location of the filtration bleb. The morphology of the bleb can be clearly observed through UBM. The filtration bleb in the hydrogel group still exists at 28 days, while the bleb in the trabecular group only lasts for 14 days, indicating that P (HEMA-CBOH) hydrogel can better maintain the existence of the bleb.

[0061] Example 11: HE staining after GFS surgery Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0062] Test method: Hematoxylin and eosin (H&E) staining of paraffin sections Test tool: BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0063] 2. The rabbits were euthanized on the 7th, 14th, and 28th days after surgery, and the eyeballs were isolated. The eye tissues were then fixed with 10% formalin to preserve their cell structure and then embedded in paraffin. Serial sections with a thickness of 3 μm were cut.

[0064] 3. Stain with hematoxylin and eosin (H&E) and observe under a microscope.

[0065] like Fig. 9 As shown, histopathological examinations were performed on the 7th, 14th and 28th days after surgery to observe changes in ocular tissues. The experimental results clearly showed that in the trabecular group, the sclera showed obvious blue-stained cell nuclei aggregation at different observation time points (marked by red arrows), which was significantly more than that in the normal control group and the hydrogel group, suggesting a stronger inflammatory response. In addition, the presence of P(HEMA-CBOH) hydrogel was still observed at various time points, which fully demonstrated its excellent biological stability.

[0066] Example 12: Masson staining after GFS surgery Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0067] Test method: Masson staining of paraffin sections Test tool: BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0068] 2. The rabbits were euthanized on the 7th, 14th, and 28th days after surgery, and the eyeballs were isolated. The eye tissues were then fixed with 10% formalin to preserve their cell structure and then embedded in paraffin. Serial sections with a thickness of 3 μm were cut.

[0069] 3. All sections were stained with Masson and observed under a microscope.

[0070] The deposition of collagen tissue is a key step in the healing process of GFS; however, excessive collagen formation may have a negative impact on surgical outcomes. Fig.10 As shown, at each observation time point after surgery, the staining in the scleral area of ​​the trabecular group was significantly deeper than that of the hydrogel group, revealing the excessive deposition of collagen tissue. When tissue is damaged or an inflammatory response occurs, the space between collagen fibers expands, resulting in loose and irregular fiber arrangement. Through comparative observation, the present invention found that the collagen fibers in the trabecular group were more sparsely arranged compared with the other two groups. The above results indicate that P (HEMA-CBOH) hydrogel can more effectively reduce collagen deposition and inflammatory response.

[0071] Example 13: Immunohistochemical staining after GFS surgery Animals and their sources: New Zealand white rabbits (2.0-2.5 kg) were purchased from Sibeifu (Beijing) Biotechnology Co., Ltd.

[0072] Test method: Immunohistochemical staining of paraffin sections (primary antibody is a-SMA, secondary antibody is HRP) Test tool: BX51 microscope (Olympus Optical Co., Ltd., Tokyo, Japan) Experimental steps: 1. New Zealand white rabbits were randomly divided into a normal control group, a trabecular group, and a hydrogel group, with 6 rabbits in each group.

[0073] 2. The rabbits were euthanized on the 7th, 14th, and 28th days after surgery, and the eyeballs were isolated. The eye tissues were then fixed with 10% formalin to preserve their cell structure and then embedded in paraffin. Serial sections with a thickness of 3 μm were cut.

[0074] 3. The primary antibody was labeled with α-SMA (Proteintech, 1:200), and the secondary antibody was labeled with horseradish peroxidase HRP (Thermo, 1:1000). After staining, the cells were observed under a microscope.

[0075] like Fig.11As shown, the present invention detected the expression level of α-SMA in rabbit eyes at different times. α-SMA is often used as a marker protein for smooth muscle cells or myofibroblasts. Myofibroblasts are a type of cell that appears during wound healing and tissue fibrosis. Compared with the normal group and the trabecular group, the expression of α-SMA in the hydrogel group was lower. This shows that P(HEMA-CBOH) hydrogel has a significant effect in preventing tissue fibrosis after GFS surgery.

[0076] Based on the above description, the present invention obtains a hydrogel that can effectively reduce and alleviate intraocular pressure after glaucoma filtration surgery and reduce scarring reaction by optimizing the selection of reactants and processes. The present invention makes full use of the good mechanical properties and biocompatibility of PHEMA, and combines it with CBOH monomer to solve the problem that PHEMA-based hydrogel still has low water content and high modulus, resulting in severe foreign body sensation, wear resistance and insufficient lubricity. Through experimental verification, it is determined that the addition amount of CBOH monomer, initiator 1173 and chemical crosslinker TEGDMA is 0.1-15wt%, 0.1-5 wt% and 0.1-1wt% of the total mass, respectively, to obtain the hydrogel of the present invention. At the same time, the optimal addition amount of the three is further determined to be 5-13 wt%, 0.5-3wt% and 0.2-0.6 wt%, so that the hydrogel of the present invention achieves the ideal technical effect. Furthermore, the present invention determines the addition amount of the three to be 12 wt%, 1 wt% and 0.5 wt% of the total mass, so that the hydrogel product achieves outstanding technical effects.

[0077] It should be further explained that the above implementation modes are only used for understanding the technical solutions of the present invention, and are not used to limit the protection scope of the present invention. Any obvious adjustments and modifications made to the technical solutions of the present invention within the technical concept of the present invention should also fall within the protection scope of the present invention.

Claims

1. A hydrogel for preventing scarring after glaucoma filtration surgery, characterized in that: The method comprises 2-hydroxyethyl methacrylate HEMA and 2-((2-hydroxy-3-(methacryloyloxy)propyl)dimethylamino)acetate CBOH, wherein the concentration of the CBOH monomer is 0.1-15wt% relative to the total mass.

2. The method for preparing the hydrogel for preventing scarring after glaucoma filtration surgery according to claim 1, characterized in that Here are the steps: 1) Heat and dissolve the CBOH monomer in the HEMA solution to form a uniform medium solution; 2) adding a photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone and a chemical crosslinker triethylene glycol dimethacrylate to the solution obtained in step 1), reacting at room temperature for 20 minutes, and mixing them thoroughly to obtain a HEMA-CBOH solution; 3) In a UV crosslinking chamber, irradiate the HEMA-CBOH solution obtained in step 2) under UV light for 1 hour to obtain P(HEMA-CBOH) hydrogel; 4) The P(HEMA-CBOH) hydrogel obtained in step 3) was immersed in 75% ethanol overnight, and then dialyzed in sterile PBS for 4-8 days to remove residual small molecules, and a sterile hydrogel was obtained after solubilization equilibrium.

3. The method for preparing a hydrogel for preventing scarring after glaucoma filtration surgery according to claim 2, characterized in that: In step 1), the mass ratio of the added CBOH monomer is 0.1-15 wt% relative to the total mass, the heating temperature is 80-100 degrees, and the heating time is 2-5 minutes.

4. The method for preparing a hydrogel for resisting scarring reaction after glaucoma filtration surgery according to claim 2, characterized in that: In step 2), the mass ratio of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.1-5wt% relative to the total mass, and the mass ratio of the chemical crosslinking agent triethylene glycol dimethacrylate is 0.1-1 wt% relative to the total mass.

5. The method for preparing a hydrogel for resisting scarring reaction after glaucoma filtration surgery according to claim 2, characterized in that: Step 3) The wavelength of UV light used is 365nm.

6. The method for preparing a hydrogel for resisting scarring reaction after glaucoma filtration surgery according to claim 2, characterized in that: After the solubilization equilibrium in step 4), the final thickness of the hydrogel obtained is 100-1000 μm.

7. The method for preparing a hydrogel for preventing scarring after glaucoma filtration surgery according to claim 3, characterized in that: The amount of CBOH monomer added is 5-13 wt% relative to the total mass.

8. The method for preparing a hydrogel for preventing scarring after glaucoma filtration surgery according to claim 4, characterized in that: The mass proportion of the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone is 0.5-5 wt % relative to the total mass, and the mass proportion of the chemical crosslinking agent triethylene glycol dimethacrylate is 0.2-0.6 wt % relative to the total mass.

9. An anti-scarring drug carrier, characterized in that: The hydrogel for resisting scarring reaction after glaucoma filtration surgery as claimed in claim 1 is used for loading anti-scarring and hormone drugs.

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