Hydrogel for inhibiting pathological neovascularization and preparation method and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a hydrogel for inhibiting pathological neovascularization and a preparation method and application thereof. The hydrogel takes methacrylated gelatin as a three-dimensional network framework, and neutralizing antibodies are embedded in the pores of the three-dimensional network framework. The neutralizing antibodies are any one of the following: a Gal3 neutralizing antibody, a combination of a Gal3 neutralizing antibody and a VEGF neutralizing antibody, and the mass ratio of the Gal3 neutralizing antibody and the VEGF neutralizing antibody is 1:0.5-2. The hydrogel is cured in situ by blue light crosslinking, and can encapsulate the Gal3 neutralizing antibody and / or the anti-VEGF antibody. After intravitreal single injection, the hydrogel can continuously release the neutralizing antibodies in the eye for at least 7 days, significantly prolongs the action time of the drug, and reduces the frequency of frequent administration. The hydrogel provides a new strategy of high efficiency, long-acting and immunoregulation for treating retinal neovascular diseases such as diabetic retinopathy, retinopathy of prematurity, age-related macular degeneration and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a hydrogel that inhibits pathological angiogenesis, its preparation method, and its application. Background Technology

[0002] Neovascular diseases of the retina (such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration) are the main causes of irreversible vision loss. Their core pathological feature is the formation of abnormal new blood vessels. These vessels are fragile, have insufficient pericyte coverage, and are dysregulated in angiogenesis signals, which can easily lead to leakage, hemorrhage, and secondary tissue damage.

[0003] Currently, anti-vascular endothelial growth factor (anti-VEGF) drugs are the first-line standard treatment for these diseases. Although they can effectively inhibit angiogenesis in clinical applications, they still have many limitations: frequent intravitreal injections are required, and repeated punctures increase the risk of intraocular infection, hemorrhage, and retinal detachment, and patient compliance is poor; significant heterogeneity in response, with patients responding poorly to anti-VEGF treatment or developing secondary resistance, resulting in limited long-term benefits; and the short half-life of the drugs leads to rapid clearance of free antibodies in the eye, making it difficult to maintain effective therapeutic concentrations. Therefore, there is an urgent need to develop a novel treatment strategy that can sustainably release neutralizing antibodies. Summary of the Invention

[0004] The purpose of this invention is to provide a hydrogel that inhibits pathological angiogenesis, thus solving the problems existing in the prior art.

[0005] The technical solution adopted in this invention is: This invention provides a hydrogel that inhibits abnormal angiogenesis, wherein the hydrogel uses methacrylamide gelatin as a three-dimensional network framework, and neutralizing antibodies are embedded in the pores of the three-dimensional network framework; The neutralizing antibody is any one of the following: (1) Gal3 neutralizing antibody; (2) A combination of Gal3 neutralizing antibody and VEGF neutralizing antibody, wherein the mass ratio of Gal3 neutralizing antibody to VEGF neutralizing antibody is 1:0.5~2.

[0006] A second aspect of the present invention provides a method for preparing the hydrogel, comprising the following steps: The photoinitiator was dissolved in a phosphate buffer solution to obtain a photoinitiator solution; Methacrylamide gelatin was dissolved in a photoinitiator solution, and after cooling to room temperature, neutralizing antibody was added to obtain the hydrogel. The mass ratio of photoinitiator to methacrylamide gelatin is 0.02~0.08:2.

[0007] Preferably, the photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite.

[0008] Preferably, when methacrylamide gelatin is dissolved in a photoinitiator solution, the photoinitiator solution needs to be heated to 65°C.

[0009] Preferably, the concentration of the neutralizing antibody in the hydrogel is 100 μg / mL.

[0010] Preferably, the process for preparing the photoinitiator solution is as follows: The photoinitiator is dissolved in PBS at 45°C to obtain the photoinitiator solution.

[0011] A third aspect of the present invention provides an application of the hydrogel, characterized in that the hydrogel is used to prepare a drug for treating retinal neovascular diseases.

[0012] The neovascular diseases of the retina include diabetic retinopathy, retinopathy of prematurity, or age-related macular degeneration.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention provides a hydrogel for inhibiting abnormal angiogenesis. The hydrogel uses methacrylamide gelatin as a three-dimensional network framework, with neutralizing antibodies embedded in the pores of the framework. The neutralizing antibody is any one of the following: (1) a Gal3 neutralizing antibody; (2) a composition of a Gal3 neutralizing antibody and a VEGF neutralizing antibody, wherein the mass ratio of the Gal3 neutralizing antibody to the VEGF neutralizing antibody is 1:0.5~2. The hydrogel of this invention is in-situ cured by blue light crosslinking, enabling the encapsulation of Gal3 neutralizing antibodies and / or VEGF antibodies. After a single intravitreal injection, the hydrogel can continuously release neutralizing antibodies into the eye for at least 7 days, significantly prolonging the duration of drug action and reducing the frequency of administration.

[0014] Compared to traditional anti-VEGF therapy, the hydrogel containing anti-Gal3 antibody in this invention not only effectively inhibits the formation of pathological neovascularization, but also uniquely inhibits the retinal recruitment of peripheral neutrophils and the release of neutrophil extracellular traps, thereby blocking the microglia-neutrophil positive feedback immune loop at its source and comprehensively improving the retinal immune microenvironment. Specifically: after a single injection of the anti-Gal3@GelMA hydrogel, the antibody can be continuously released until day 7, while the free antibody in the control group significantly decays on day 2; the hydrogel effectively reduces the density of retinal neovascularization and the area of ​​non-perfusion areas, with efficacy comparable to anti-VEGF, and the effect is even better when anti-Gal3 and anti-VEGF are used in combination; the anti-Gal3 neutralizing antibody can downregulate the TLR4 / TRIF / caspase-8 signaling pathway, reduce neutrophil NETs formation, and reduce the level of retinal inflammatory factors, a mechanism of action that anti-VEGF drugs do not possess; the hydrogel is completely cleared in vivo on day 35, showing good biocompatibility and safety.

[0015] Therefore, the hydrogel of the present invention provides a novel strategy with high efficiency, long-lasting effect and immunomodulatory function for the treatment of retinal neovascular diseases such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration. Attached Figure Description

[0016] Figure 1 This describes the preparation process of hydrogels.

[0017] A: Schematic diagram of hydrogel preparation; B: Photographs of the hydrogel before and after irradiation with 405nm ultraviolet light.

[0018] Figure 2 Image of hematoxylin-eosin (HE) staining on a cross-section of a mouse retina.

[0019] Figure 3 To compare the intraocular retention time of hydrogel-encapsulated Gal3 neutralizing antibodies versus Gal3 neutralizing antibodies alone after retinal injection; A: In vivo imaging system diagram of adult mice after intravitreal injection of Gal3 neutralizing antibody alone; B: In vivo imaging system diagram of adult mice after intravitreal injection of hydrogel-encapsulated Gal3 neutralizing antibodies; The quantitative graph of fluorescence intensity in C:B was normalized based on the fluorescence intensity at 0 hours post-injection.

[0020] Figure 4 The duration of intraocular retention of Gal3 neutralizing antibody conjugated with AF488 encapsulated in hydrogel and attached to the retina after injection.

[0021] A: Observe the intraretinal fluorescence of adult mice at different time points after intravitreal injection of AF488-labeled AbGAL3.

[0022] Figure 5 To compare the changes in intraocular inflammation after injection of hydrogel-encapsulated Gal3 neutralizing antibodies versus hydrogel-only retinal injection; A: Immunofluorescence staining image, retinal neutrophils (LY6G, red) and nuclei (DAPI, blue) in cross-sections of the retina of mice in the NC@GelMA group and the AbGAL3@GelMA group after GelMA injection. Scale bar: 20µm;

[0023] B: Immunofluorescence staining image, retinal NETs (CitH3, purple) and cell nuclei (DAPI, blue) in cross-sections of the retina of mice in the NC@GelMA group and the AbGAL3@GelMA group after GelMA injection. Scale bar: 20µm;

[0024] C: Western blot image, showing the expression of IL-6 and IL-1β in the retina after intravitreal injection in the NC@GelMA group and the AbGAL3@GelMA group.

[0025] Figure 6 To compare the changes in intraocular inflammation after retinal injection of simple hydrogel, hydrogel-encapsulated VEGF neutralizing antibody, hydrogel-encapsulated Gal3 neutralizing antibody, and hydrogel simultaneously encapsulating Gal3+VEGF neutralizing antibody; A: Immunofluorescence staining of retinal vessels (CD31, red) in OIR group mice at P17; B: Dark red indicates neovascularization; C: Yellow indicates VO vascular occlusion. Scale bar: 500µm;

[0026] D: Immunofluorescence staining of retinal vessels (CD31, red) in mice in the hydrogel-only injection group at P17; E: Dark red indicates neovascularization; F: Yellow indicates VO vascular occlusion. Scale bar: 500µm;

[0027] G: Immunofluorescence stained section of retinal vessels (CD31, red) in the VEGF neutralizing antibody-injected mice encapsulated in hydrogel at P17; H: Dark red indicates neovascularization; I: Yellow indicates VO vascular occlusion. Scale bar: 500µm;

[0028] J: Immunofluorescence stained section of retinal vessels (CD31, red) in mice injected with GAL3 neutralizing antibody and encapsulated in hydrogel at P17; K: Dark red indicates neovascularization; L: Yellow indicates VO occlusion. Scale bar: 500µm;

[0029] M: Immunofluorescence stained section of retinal vessels (CD31, red) in mice injected with Gal3+VEGF neutralizing antibody and encapsulated in hydrogel at P17; N: Dark red indicates neovascularization; O: Yellow indicates VO vascular occlusion. Scale bar: 500µm;

[0030] P: Quantification of neovascularization area in A, D, G, J and M; Q: Quantification of the areas of non-irrigated zones A, D, G, J, and M.

[0031] Figure 7 The study analyzed the spontaneous degradation of the hydrogel within the vitreous cavity. Hematoxylin-eosin (HE) staining images of retinal cross sections on days 1, 7, 14, 21, 28, and 35 after injection of the hydrogel containing neutralizing antibodies into the vitreous cavity of C57 mice were also included. Detailed Implementation

[0032] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0033] Preliminary research for this invention revealed: 1. Patients with retinal neovascularization disease exhibit retinal immune microenvironment dysregulation and peripheral neutrophil recruitment.

[0034] To investigate immune mobilization in retinal vascular diseases, single-cell RNA sequencing (scRNA-seq) data (Series GSE165784) of fibrovascular membranes (FVMs) from patients with advanced diabetes mellitus in the GEO database were analyzed. Abundant myeloid cell populations were also observed in the FVM samples, indicating peripheral immune cell infiltration into the retinal tissue. To systematically explore this peripheral immune involvement, blood samples were collected from 12 patients with proliferative diabetic retinopathy (DR) and 12 patients with idiopathic epiretinal membranes. Comparative analysis of peripheral blood leukocyte profiles showed a significant increase in total white blood cell count, with the most significant increase in neutrophils. Bivariate logistic regression analysis showed that among neutrophil, monocyte, and lymphocyte percentages, only the neutrophil percentage was an independent risk factor for DR development. These findings suggest that systemic immune alterations and neutrophil involvement may be related to retinal vascular pathology. Immunofluorescence staining further confirmed neutrophil activation within FVMs and the involvement of resident mycoplasma in the retina, indicating that neutrophils are potential contributors to the pathogenesis of DR. These findings suggest that peripheral neutrophil mobilization, along with MG activation, disrupts the retinal immune microenvironment in retinal vascular diseases.

[0035] 2. The mutual regulation between microglia and neutrophils synergistically disrupts the retinal immune microenvironment.

[0036] To elucidate the role of neutrophils in the retinal microenvironment, this invention used single-cell RNA sequencing to examine intercellular communication between major retinal cell types and neutrophils. Analysis revealed extensive interactions not only between neutrophils themselves but also between neutrophils and MGs. However, it remained unclear which cell type initiated this pathological response. To address this issue, the number and activation status of MGs and neutrophils in the retinas of OIR mice from P12 to P17 were assessed. By P13, MGs exhibited activated amoeboid morphology, characterized by enlarged cell bodies and contracted processes. However, neutrophils were not detected until P15, indicating that MG activation preceded neutrophil infiltration.

[0037] 3. Gal3 derived from microglia triggers neutrophil infiltration and NET formation.

[0038] To elucidate the molecular mechanisms by which MG regulates neutrophils, single-cell RNA sequencing was performed on MG in the OIR retina. The results showed that Lgals3, encoding galectin-3 (Gal3), was the most significantly upregulated gene in MG. Consistently, Gal3 expression was also upregulated in the vitreous and aqueous humor of patients with proliferative diabetic retinopathy (DR) compared to normal controls. IF staining confirmed increased Gal3 expression in OIR mice. Inhibition of Gal3 by intravitreal injection of a Gal3-neutralizing antibody effectively reduced retinal angiogenesis. Compared to anti-VEGF injection, anti-Gal3 injection showed comparable efficacy in inhibiting angiogenesis. Furthermore, when comparing the effects of anti-Gal3 and anti-VEGF on neutrophil involvement, NET formation, and inflammation, anti-Gal3 exhibited a unique inhibitory capacity, which was almost unobservable with anti-VEGF treatment, suggesting that Gal3 may promote angiogenesis through a mechanism different from the VEGF pathway. Since MG primarily expresses Gal3, we constructed and analyzed MG-specific Gal3 knockout mice. After establishing the OIR model, Gal3 KO mice showed significantly reduced neutrophil infiltration and NET formation, along with decreased expression of related molecular markers. In contrast, when Gal3 KO mice received intravitreal injections of Gal3-expressing BV2 cells, neutrophil recruitment to neovascularization sites was restored, and pathological angiogenesis was exacerbated. These findings suggest that MG-derived Gal3 promotes neutrophil aggregation and NET formation, thereby promoting retinal angiogenesis. To confirm the neutrophil-microglia-Gal3 axis observed in OIR, evaluation was conducted using high-fat diet (HFD) and streptozotocin (STZ) induced diabetes models, as well as a db / db diabetes model. Gal3 was found to regulate neutrophil infiltration, thereby modulating NET formation.

[0039] 4. The Gal3-TLR4 pathway promotes the release of NETs.

[0040] Next, we investigated the mechanism by which Gal3 regulates NET formation. Gene expression analysis of neutrophils before and after establishing the OIR model showed that, in addition to neutrophil-specific markers, the expression of Toll-like receptor 4 (TLR4) was significantly upregulated. Notably, intravitreal injection of anti-Gal3 neutralizing antibodies in OIR mice downregulated the expression of TLR4 and its adaptor protein TRIF, and also reduced caspase-8 expression. Furthermore, primary neutrophils isolated from mouse peripheral blood samples showed significantly increased expression of TLR4, TRIF, and caspase-8 after Gal3 stimulation, indicating that Gal3 may trigger downstream signaling through TLR4. Molecular docking models and immunoprecipitation (IP) analysis supported this hypothesis, confirming a direct interaction between Gal3 and TLR4. IF co-localization analysis further showed that retinal Gal3 blockade effectively inhibited the TLR4 pathway, thereby reducing NET formation. To explore the functional role of TLR4, we injected the TLR4 inhibitor TAK-242 and its agonist neoseptin intravitreally. Gal3 inhibition significantly attenuated retinal neovascularization and vascular occlusion, while activation of TLR4 via neoseptin and Gal3 supplementation exacerbated these pathological features. Conversely, TLR4 inhibition reversed these effects. In vivo, TLR4 inhibition significantly reduced NET production. In vitro, TAK-242 blocked Gal3-induced NET formation, while neoseptin restored it, highlighting the crucial role of the Gal3-TLR4 pathway in NET formation.

[0041] 5. Neutrophil-derived NETs amplify MG activation, forming a positive feedback loop with MG.

[0042] To further explore the regulatory interactions among neutrophils, NETs, ​​and MG, we established a co-culture system combining BV2 MG cells with primary peripheral blood neutrophils and PMA-induced NETs. Batch RNA sequencing of BV2 cells revealed significant transcriptomic alterations in different treatment groups compared to the control group. Gene ontology (GO) enrichment analysis highlighted key changes in immune responses, cell migration and adhesion, angiogenesis-related processes, and ligand-receptor interactions. We also examined the expression of MG activation markers Itgax, Lgals3, CD9, Itgam, Apoe, and CX3CR1. Notably, these genes were highly upregulated in the NET-containing co-culture group but downregulated in the neutrophil-only co-culture group, highlighting the crucial role of NETs in MG activation. Pairwise pathway enrichment analysis showed that co-culturing with neutrophils only altered major cell component-related pathways compared to the BV2 monoculture control. In contrast, BV2 cells co-cultured with neutrophils that induce NET formation exhibited phenotypic changes consistent with immune activation and pro-angiogenic MG. Intravitreal injection of the NET inhibitor GSK484 into OIR mice revealed altered MG localization: in untreated OIR retinas, activated MG migrated to superficial lesion areas, while NET inhibition confined MG to the outer plexiform layer, indicating suppressed activation. Similarly, additional intravitreal injection of purified NETs extracted from stimulated neutrophils into the eyes of newborn mice induced altered microglial morphology, further demonstrating that NETs induce MG activation. The expression of Lgals3 (encoding gal-3), pro-angiogenic factor (Vegfa), and angiogenesis and inflammation-related factors (Ang2 / Ang1 and Tnf-α) were all significantly upregulated. Morphological analysis of BV2 cells stimulated with NETs derived from primary neutrophils under in vitro conditions showed a significant increase in aspect ratio and nuclear area, indicating cell activation. Notably, exposure to neutrophil-derived NETs elevated Gal3 expression in both BV2 cells and their supernatant, suggesting that Gal3 may, in turn, promote further NET formation, creating an amplified feedback loop. Furthermore, PMA-induced NET supernatant significantly upregulated Lgals3, neutrophil chemotaxis-related genes (Cxcl1 and Cxcl2), Vegfa, Ang2 / Ang1, and Tnf-α in BV2 cells. In summary, these findings demonstrate that NETs enhance MG activation, triggering a Gal3-dependent cascade that enhances neutrophil recruitment and pathological angiogenesis. These data further support a self-reinforcing cycle between MG and neutrophils, where mutual activation amplifies Gal3 accumulation and NET formation, thereby exacerbating disease progression.

[0043] Based on this, the present invention provides a hydrogel that inhibits pathological angiogenesis, the hydrogel comprising methacrylamide gelatin, a photoinitiator, and a neutralizing antibody. The inventive concept of the present invention is as follows:

[0044] 1. GelMA (methacrylamide gelatin) - skeleton material.

[0045] Function: Serves as the basic framework for photocrosslinked hydrogels. It is obtained by modifying gelatin with methacrylamide groups, thus retaining gelatin's good biocompatibility, cell adhesion sites (RGD sequence), and degradability.

[0046] Its role in this system: After dissolution, it forms a polymer network precursor. Under light irradiation, the double bonds on its molecular chains cross-link, transforming it from a liquid state into a solid hydrogel, which serves as a physical support and encapsulates the antibody.

[0047] 2. LAP (Lithium Phenyl-2,4,6-trimethylbenzoylphosphite) - Photoinitiator.

[0048] Function: It acts as a "crosslinking switch". It is a water-soluble, cell-compatible photoinitiator that decomposes to produce free radicals when irradiated with specific wavelengths (usually 405nm blue light or 365nm ultraviolet light).

[0049] Its role in this system: It does not form a gel itself, but rather triggers the cross-linking reaction of GelMA. Upon light exposure, the free radicals generated by LAP induce the polymerization of double bonds on the GelMA molecular chains, causing the solution to rapidly solidify into a gel. Without LAP, the GelMA solution cannot be photocured.

[0050] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below with reference to specific embodiments. In the description of the present invention, unless otherwise specified, all reagents used are commercially available, and all methods used are conventional techniques in the art.

[0051] The materials used in this invention are sourced from the following sources: GelMA was purchased from EFL-GM-60; Gal3 neutralizing antibody: GeneTex, Cat# GTX41041; VEGF neutralizing antibody: R&D, Cat# AF-493-NA.

[0052] The list of abbreviations for this invention is shown in Table 1.

[0053] Table 1 List of Abbreviations Example 1 A hydrogel that inhibits pathological angiogenesis, the specific preparation method of which is as follows: Dissolve 0.05 g LAP in 20 mL PBS at 45°C to obtain the LAP solution.

[0054] Dissolve 2g of GelMA in LAP solution at 65°C to 10% (w / v), cool to room temperature, add Gal3 neutralizing antibody to make the final concentration of Gal3 neutralizing antibody reach 100μg / mL, mix well, and the hydrogel is obtained, denoted as @GelMA.

[0055] During in vivo injection, the composite hydrogel was cross-linked using 405nm blue light for 30 seconds. After blue light irradiation, the hydrogel changed from a liquid to a solid state. Figure 1 A and B.

[0056] Example 2 A hydrogel that inhibits pathological angiogenesis, the specific preparation method of which is as follows: Dissolve 0.05 g LAP in 20 mL PBS at 45°C to obtain the LAP solution.

[0057] Dissolve 2g of GelMA in LAP solution at 65°C to 10% (w / v), cool to room temperature, add Gal3 neutralizing antibody and VEGF neutralizing antibody to make the final concentration of the two antibodies reach 100μg / mL, mix well, and the hydrogel is obtained.

[0058] During in vivo injection, the composite hydrogel was cross-linked using 405nm blue light for 30 seconds. After irradiation with blue light, the hydrogel changed from a liquid state to a solid state. The products prepared in Examples 1 and 2 were cross-linked with blue light for 30 seconds, freeze-dried, and longitudinally sliced. The hydrogel samples with different components were sputtered with gold under vacuum and observed to have a network porous structure under a scanning electron microscope (Thermo Fisher Scientific Quattro S, USA).

[0059] Example 3 The application of a hydrogel that inhibits pathological angiogenesis was verified by examining the effects of the hydrogels prepared in Examples 1 and 2, as detailed below: 1. Experimental method - intravitreal injection.

[0060] C57BL / 6J mice were anesthetized via intraperitoneal injection. Topical tropicamide (1%) was used for mydriasis. Under a surgical microscope, using a 33-gauge Hamilton syringe, 1 µL of liquid hydrogel (containing 100 µg of fluorescently labeled protein AF488 per µL) was injected into the vitreous cavity through the pars plana of the ciliary body, 1 mm posterior to the limbus. Irradiation with blue light caused the hydrogel to solidify and adhere to the retinal surface. Lens damage was avoided during injection. The contralateral eye received the fluorescently labeled protein as a control. Antibiotic ointment was applied post-injection.

[0061] 2. Experimental results.

[0062] Given the central role of Gal3 in maintaining immune dysregulation, this invention aims to disrupt this positive feedback loop by targeting MG-derived Gal3, thereby reducing angiogenesis. To this end, Examples 1 and 2 of this invention develop a photocurable hydrogel capable of continuously releasing anti-Gal3 neutralizing antibodies after a single intravitreal injection.

[0063] Irradiation with a 405 nm light source resulted in the solidification of the hydrogel, and cross-linking with the antibody did not significantly alter its pore size or elastic modulus. The hydrogel precursor was injected intravitreally, followed by in-situ photopolymerization. H&E staining confirmed that the hydrogel adhered tightly to the retinal surface. Results are shown in [Figure number missing]. Figure 2 .

[0064] This invention conjugates anti-Gal3 with the AF488 fluorophore and encapsulates it in a hydrogel (AbGal3@GelMA). The complex was injected into the vitreous humor of mice via a serrated needle and irradiated with blue light. Fluorescence intensity was monitored using an in vivo imaging system. It was observed that the fluorescence intensity decreased on day 2 after injection of the Gal3-only neutralizing antibody, while the hydrogel-encapsulated neutralizing antibody persisted until day 10. Figure 3 and Figure 4 Furthermore, the hydrogels attached to the inner retina were separated at different time points, and the fluorescence intensity was quantified by confocal imaging to calculate the cumulative release curve of the antibodies encapsulated in the hydrogels. Figure 3 The C-value suggests that the neutralizing antibody can be slowly released up to day 10 after hydrogel encapsulation. Simultaneously, it was observed that the fluorophore-labeled antibody reached the retina on the second day after injection to exert its therapeutic effect, and its fluorescence morphology was similar to that of microglia. Furthermore, this delivery system was found to effectively inhibit the accumulation of neutrophils and NETs. Figure 5 (A and B) and the level of inflammation in the retina ( Figure 5 (C).

[0065] Furthermore, by comparing intraocular injection of NC@GelMA alone, anti-VEGF AbVEGF@GelMA, anti-Gal3 AbGal3@GelMA, and combined anti-Gal3 and anti-VEGF treatments in OIR ( Figure 6 After treatment, the area of ​​neovascularization decreased, with the most significant reduction observed with the combination of V+G therapy; the area of ​​non-perfusion zone also decreased significantly, again with the most significant reduction observed with the combination of V+G therapy. Figure 6 G). The treatment groups are shown in Table 2.

[0066] Table 2. Dosage for different treatment groups Anti-Gal3@GelMA effectively reduces angiogenesis density and non-perfusion area; combined application shows even better inhibitory effects on both angiogenesis and avascular areas. Furthermore, this invention demonstrated that the hydrogel was completely removed by day 35, without the need for a second surgery, showing significant potential for translational applications. Figure 7 In summary, these results highlight the promising therapeutic approach of delivering hydrogel-encapsulated Gal3-neutralizing antibodies intravitreally as a target of the Gal3-driven microglia-NET feedback loop and its amplified cascade.

[0067] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0068] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A hydrogel that inhibits abnormal angiogenesis, characterized in that, The hydrogel uses methacrylamide gelatin as a three-dimensional network framework, with neutralizing antibodies embedded in the pores of the three-dimensional network framework. The neutralizing antibody is any one of the following: (1) Gal3 neutralizing antibody; (2) A combination of Gal3 neutralizing antibody and VEGF neutralizing antibody, wherein the mass ratio of Gal3 neutralizing antibody to VEGF neutralizing antibody is 1:0.5~2.

2. The method for preparing the hydrogel according to claim 1, characterized in that, The steps are as follows: The photoinitiator was dissolved in a phosphate buffer solution to obtain a photoinitiator solution; Methacrylamide gelatin was dissolved in a photoinitiator solution, and after cooling to room temperature, neutralizing antibody was added to obtain the hydrogel. The mass ratio of photoinitiator to methacrylamide gelatin is 0.02~0.08:

2.

3. The preparation method according to claim 2, characterized in that, The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoyl phosphite.

4. The preparation method according to claim 2, characterized in that, When methacrylamide gelatin is dissolved in a photoinitiator solution, the photoinitiator solution needs to be heated to 65°C.

5. The preparation method according to claim 2, characterized in that, The concentration of the neutralizing antibody in the hydrogel is 100 μg / mL.

6. The preparation method according to claim 2, characterized in that, The process for preparing the photoinitiator solution is as follows: The photoinitiator is dissolved in PBS at 45°C to obtain the photoinitiator solution.

7. The application of the hydrogel as described in claim 1, characterized in that, The hydrogel is used to prepare drugs for the treatment of retinal neovascularization diseases.

8. The application as described in claim 7, characterized in that, The neovascular diseases of the retina include diabetic retinopathy, retinopathy of prematurity, or age-related macular degeneration.

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