Gelatin-sodium alginate / conbercept composite gel modified cross-linked amnion and its preparation method

Abstract

This invention relates to the fields of biomedical materials and ophthalmology, specifically disclosing a gelatin-sodium alginate / conbercept composite gel-modified crosslinked amniotic membrane and its preparation method. The composite gel-modified crosslinked amniotic membrane comprises a crosslinked amniotic membrane and a gelatin-sodium alginate / conbercept composite gel layer bonded to the surface of the crosslinked amniotic membrane. The composite gel layer is formed by calcium ion crosslinking of a gelatin-sodium alginate composite hydrogel solution containing conbercept. The preparation method includes amniotic membrane pretreatment, riboflavin / ultraviolet crosslinking, gelatin-sodium alginate composite hydrogel preparation, conbercept loading, and calcium ion crosslinking molding steps. This invention improves the loading and retention capacity of conbercept on the crosslinked amniotic membrane, achieving sustained in vitro release of conbercept, and can be used as a sustained-release drug delivery material for conbercept on the ocular surface.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and ophthalmology, specifically to a gelatin-sodium alginate / conbercept composite gel modified crosslinked amniotic membrane and its preparation method. Background Technology

[0002] Corneal neovascularization (CoNV) is a common eye disease that seriously threatens vision, with an estimated 1.4 million cases annually. It is mostly secondary to pathological damage such as infection, chemical burns, and transplant rejection. The formation of corneal neovascularization can lead to decreased vision or even blindness due to edema, persistent inflammation, protein and lipid deposition in the stroma, and scarring.

[0003] Currently, clinical treatments for corneal neovascularization mainly include drug therapy (such as glucocorticoids and anti-VEGF drugs), photodynamic therapy, and corneal transplantation. However, existing treatments still have many limitations: topical eye drops have low bioavailability and require frequent administration; subconjunctival injections are invasive and have poor patient compliance; corneal transplantation surgery faces risks such as limited donor availability and transplant rejection. The application of anti-VEGF drugs provides a new option for the treatment of corneal neovascularization, but the problems of short residence time on the ocular surface and low bioavailability have not yet been effectively solved.

[0004] Human amniotic membrane (AM) is rich in various active ingredients and possesses anti-inflammatory, anti-fibrotic, and anti-angiogenic properties, making it widely used in ocular surface reconstruction surgery. Accumulated evidence shows that AM can also serve as a drug delivery carrier, loading drug molecules for slow release. However, AM is prone to gradual dissolution in vivo; Spoerl et al. reported that fresh and frozen AM can be completely degraded within approximately one week, primarily due to enzymatic degradation of its matrix. For patients with chemical corneal burns, multiple amniotic membrane transplants are often required; therefore, prolonging the retention time of AM while simultaneously releasing anti-angiogenic drugs remains a pressing challenge.

[0005] The drug loading capacity of amniotic membrane is very limited when relying solely on physical immersion methods. Literature reports that the encapsulation rate of cefazolin in amniotic membrane is only 11% to 24%. This low drug loading efficiency limits the clinical application potential of drug-loaded amniotic membrane alone.

[0006] Both gelatin and sodium alginate are naturally derived biomaterials with good biocompatibility and biodegradability. Gelatin, mainly composed of glycine-proline-hydroxyproline repeating sequences, possesses excellent biocompatibility, biodegradability, and low immunogenicity, but its individual mechanical strength and stability are relatively poor. Sodium alginate, as a natural polysaccharide, can form a three-dimensional network structure through ionic cross-linking. The gelatin-sodium alginate hydrogel prepared by combining the two not only improves mechanical properties but also has a sustained-release function. Currently, this composite system has been successfully used to construct corneal repair scaffolds and drug delivery carriers, fully demonstrating its application potential in ocular surface drug delivery and corneal repair. However, there are currently no reports on the use of gelatin-sodium alginate hydrogel combined with cross-linked amniotic membrane for sustained-release delivery of Conbercept on the ocular surface. Summary of the Invention

[0007] This invention addresses the problems of low drug loading efficiency and rapid degradation of amniotic membrane in existing technologies, as well as the short residence time and low bioavailability of anti-VEGF drugs on the ocular surface. It provides a gelatin-sodium alginate / conbercept composite gel modified crosslinked amniotic membrane for corneal neovascularization treatment and its preparation method.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: Gelatin-sodium alginate / conbercept composite gel modifies cross-linked amniotic membrane, comprising cross-linked amniotic membrane and a gelatin-sodium alginate / conbercept composite gel layer bound to the surface of cross-linked amniotic membrane; the gelatin-sodium alginate / conbercept composite gel layer is formed by calcium ion cross-linking of a gelatin-sodium alginate composite hydrogel solution containing conbercept and covers the surface of cross-linked amniotic membrane.

[0009] Cross-linked amnion is amnion that has been cross-linked with riboflavin and ultraviolet light.

[0010] The gelatin-sodium alginate composite hydrogel solution is prepared by dissolving 4% (w / v) gelatin and 2% (w / v) sodium alginate in deionized water.

[0011] The gelatin-sodium alginate / combercept composite gel layer is formed by cross-linking with calcium chloride solution.

[0012] The cross-linked amniotic membrane was a circular amniotic membrane sheet with a diameter of 15 mm; when preparing each gelatin-sodium alginate / conbercept composite gel modified cross-linked amniotic membrane sheet, the dosage of conbercept was 400 μg.

[0013] Furthermore, the present invention provides a method for preparing a cross-linked amnion modified with a gelatin-sodium alginate / conbercept composite gel, comprising the following steps: S1. The amnion is pretreated and then cross-linked with riboflavin and ultraviolet light to obtain a cross-linked amnion. S2. Add gelatin and sodium alginate to deionized water, heat and stir to dissolve, and obtain gelatin-sodium alginate composite hydrogel solution. S3. Add conbercept to the gelatin-sodium alginate composite hydrogel solution and mix well to obtain gelatin-sodium alginate / conbercept composite gel. S4. The gelatin-sodium alginate / conbercept composite gel was loaded onto the surface of the cross-linked amnion and subjected to calcium ion cross-linking treatment to obtain the gelatin-sodium alginate / conbercept composite gel modified cross-linked amnion.

[0014] In S1, the pretreated amnion was completely immersed in a 0.1% riboflavin solution and incubated for 10 minutes. Then, it was subjected to crosslinking treatment by vertical irradiation with 365nm ultraviolet light for 4 minutes. The ultraviolet light irradiation intensity was 30mW / cm², and the total irradiation energy was 7.2J / cm².

[0015] In S4, gelatin-sodium alginate / conbercept composite gel was dropped onto the surface of the cross-linked amnion and incubated at 4°C for 30 min, followed by cross-linking at 4°C for 1 min using 3% (w / v) calcium chloride solution.

[0016] The gelatin-sodium alginate / conbercept composite gel modified cross-linked amniotic membrane of the present invention can be used as a sustained-release drug delivery material for conbercept on the ocular surface, providing a material basis for the continuous local delivery of anti-vascular endothelial growth factor drugs on the ocular surface.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention modifies cross-linked amniotic membrane with gelatin-sodium alginate hydrogel and loads conbercept into the gelatin-sodium alginate composite hydrogel, thereby improving the loading and retention capacity of conbercept on the cross-linked amniotic membrane. Encapsulation efficiency measurements show that the encapsulation efficiency of the cross-linked amniotic membrane modified with the gelatin-sodium alginate / conbercept composite gel is higher than that of the cross-linked amniotic membrane loaded with conbercept.

[0018] This invention employs riboflavin and ultraviolet light to cross-link the amniotic membrane, thereby improving the density and stability of the amniotic membrane matrix structure. In vitro collagenase degradation experiments show that, compared to ordinary amniotic membrane, the cross-linked amniotic membrane exhibits stronger resistance to collagenase degradation, which is beneficial for prolonging the survival time of the amniotic membrane material in the ocular surface environment.

[0019] This invention incorporates a gelatin-sodium alginate / conbercept composite gel onto the surface of cross-linked amniotic membrane, enabling the cross-linked amniotic membrane to provide both ocular surface coverage and support, as well as sustained-release delivery of conbercept. In vitro release experiments showed that the cumulative release of conbercept from the gelatin-sodium alginate / conbercept composite gel-modified cross-linked amniotic membrane was higher than that from the conbercept-loaded cross-linked amniotic membrane.

[0020] The preparation method of this invention includes amniotic membrane pretreatment, riboflavin / ultraviolet crosslinking, preparation of gelatin-sodium alginate composite hydrogel, conbercept loading, and calcium ion crosslinking molding steps. The process conditions are mild and suitable for preparing conbercept ocular sustained-release drug-loaded materials. Attached Figure Description

[0021] Figure 1 The images show the ultrastructure of ordinary amnion and cross-linked amnion under a transmission electron microscope, where A is the ordinary amnion and B is the cross-linked amnion. Figure 2 Comparative images of the appearance of Gel-SA hydrogel and Gel-SA@CAM composite gel, where C is Gel-SA hydrogel and D is Gel-SA@CAM composite gel; Figure 3 A bar chart comparing the encapsulation efficiency of cross-linked amniotic membrane loaded with conbercept and cross-linked amniotic membrane modified with gelatin-sodium alginate / conbercept composite gel; Figure 4 A bar chart comparing the swelling rates of gelatin-sodium alginate hydrogel-modified cross-linked amniotic membrane and gelatin-sodium alginate hydrogel. Figure 5 The graph shows the results of the in vitro drug release experiment, where A is the daily release curve, B is the cumulative release curve, and C is the bar chart comparing the cumulative release on day 5. Figure 6 This graph shows the changes in the in vitro collagenase degradation rate between ordinary amnion and cross-linked amnion. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. Equivalent substitutions or simple modifications made by those skilled in the art to certain technical features based on an understanding of the technical solution of the present invention are all within the protection scope of the present invention.

[0023] Example 1: Preparation of cross-linked amnion modified with gelatin-sodium alginate / conbercept composite gel 1. Pretreatment of the amnion Placentas were harvested from healthy mothers via cesarean section. Serological tests for HBsAg, HIV, HBV, HCV, and syphilis were all negative in the donors. Amniotic membranes were collected under aseptic conditions. After rinsing with sterile saline, the membranes were incubated in sterile saline containing gentamicin (40 μg / mL) for 15 minutes, followed by rinsing three times with sterile saline to remove residual antibiotics, yielding pretreated amniotic membranes. Pretreated amniotic membranes are the same as ordinary amniotic membranes, denoted as AM.

[0024] 2. Preparation of cross-linked amnion Pretreated amniotic membrane was completely immersed in a 0.1% riboflavin solution (Sigma-Aldrich, USA) and incubated for 10 minutes to allow the riboflavin to fully penetrate the amniotic tissue. The amniotic membrane was then cross-linked using an Ovedro accelerated cross-linking system. The irradiation wavelength was 365 nm, the irradiation intensity was 30 mW / cm², the irradiation time was 4 minutes, and the total irradiation energy was 7.2 J / cm². The amniotic membrane was irradiated vertically throughout the process, resulting in a cross-linked amniotic membrane, denoted as CAM, which was stored at 4°C for later use.

[0025] 3. Preparation of gelatin-sodium alginate composite hydrogel solution Weigh 4g of gelatin and 2g of sodium alginate separately, dissolve them together in 100mL of deionized water, heat and stir continuously in a 55℃ water bath until the solid powder is completely dissolved, and prepare a 4% (w / v) gelatin-2% (w / v) sodium alginate composite hydrogel solution, denoted as Gel-SA.

[0026] The gelatin-sodium alginate composite hydrogel solution and Gel-SA mentioned below refer to the above-mentioned 4% (w / v) gelatin-2% (w / v) sodium alginate composite hydrogel solution.

[0027] 4. Preparation of gelatin-sodium alginate / conbercept composite gel The conbercept stock solution (10 mg / mL) was mixed with the gelatin-sodium alginate composite hydrogel solution (Gel-SA) at a volume ratio of 1:4. The mixture was gently blown and aspirated to obtain the gelatin-sodium alginate / conbercept composite gel, denoted as Gel-SA / Conbercept, and stored at 37°C for later use.

[0028] 5. Preparation of cross-linked amnion modified with gelatin-sodium alginate / conbercept composite gel Take the cross-linked amniotic membrane (CAM), absorb the surface moisture with sterile gauze, cut it into uniform circular pieces using a 15mm corneal trephine, and lay them flat in a 15mm diameter circular silicone mold.

[0029] The prepared gelatin-sodium alginate / conbercept composite gel (Gel-SA / Conbercept) was uniformly dropped onto the surface of each cut circular cross-linked amniotic membrane (CAM) patch, with an addition volume of 200 μL, ensuring that the gelatin-sodium alginate / conbercept composite gel solution (Gel-SA / Conbercept) covered and adhered to the surface of the cross-linked amniotic membrane (CAM). Specifically, for each circular cross-linked amniotic membrane patch, the amount of conbercept stock solution used was 40 μL, the amount of gelatin-sodium alginate composite hydrogel solution (Gel-SA) was 160 μL, and the amount of conbercept added was 400 μg. The patches were then incubated at 4 °C for 30 min to allow for thorough bonding between the gelatin-sodium alginate / conbercept composite gel (Gel-SA / Conbercept) and the cross-linked amniotic membrane (CAM). The circular amniotic membrane patches were then completely immersed in a 3% (w / v) calcium chloride solution and cross-linked at 4 °C for 1 min.

[0030] After cross-linking, the circular amniotic membrane samples were immediately rinsed three times with sterile physiological saline, and the rinsing solution was collected for later use. The rinsed circular amniotic membranes are the gelatin-sodium alginate / conbercept composite gel-modified cross-linked amniotic membranes prepared in this embodiment, denoted as Gel-SA / Conbercept@CAM.

[0031] Example 2: Preparation of control material Gelatin-sodium alginate hydrogel-modified cross-linked amniotic membrane without conbercept and conbercept-loaded cross-linked amniotic membrane without gelatin-sodium alginate hydrogel were prepared as control materials for subsequent experiments.

[0032] Comparative Example 1: Preparation of cross-linked amnion modified with gelatin-sodium alginate hydrogel The cross-linked amniotic membrane (CAM) prepared in Example 1 was dried with sterile gauze and cut into circular pieces using a 15mm corneal trephine. The circular amniotic membrane pieces were then laid flat in a small petri dish. A gelatin-sodium alginate composite hydrogel solution (Gel-SA) was uniformly dropped onto the surface of the circular amniotic membrane pieces, ensuring the Gel-SA covered the cross-linked amniotic membrane. The mixture was incubated at 4°C for 30 min. After incubation, the surface of the circular amniotic membrane pieces was gently rinsed with sterile physiological saline, followed by complete immersion in a 3% (w / v) calcium chloride solution. Cross-linking was then performed at 4°C for 1 min. Immediately after cross-linking, the circular amniotic membrane pieces were rinsed three times with sterile physiological saline, and the rinsing solution was collected for later use. The rinsed amniotic membrane is the gelatin-sodium alginate hydrogel-modified cross-linked amniotic membrane, denoted as Gel-SA@CAM.

[0033] Comparative Example 2: Preparation of Conbercept-crosslinked amnion The cross-linked amniotic membrane (CAM) prepared in Example 1 was blotted dry with sterile gauze and cut into circular pieces using a 15mm corneal trephine. The circular amniotic membrane pieces were then laid flat in a small petri dish. 40 μL of sterile physiological saline containing 400 μg of conbercept was added to the surface of the circular amniotic membrane pieces, ensuring full contact between the conbercept solution and the cross-linked amniotic membrane. The mixture was incubated at 4°C for 30 min. After incubation, the circular amniotic membrane pieces were removed and gently rinsed three times with sterile physiological saline. The rinsing solution was collected for later use. The rinsed amniotic membrane is the conbercept-loaded cross-linked amniotic membrane, denoted as CAM / Conbercept.

[0034] For ease of differentiation and reference in subsequent embodiments, the names and abbreviations of the preparations in Examples 1-2 are shown in the table below.

[0035] Table 1. Explanation of each preparation and abbreviation Example 3: Determination of encapsulation efficiency of cross-linked amniotic membrane modified with gelatin-sodium alginate / conbercept composite gel In Example 1, the rinsing solution generated by washing with sterile saline during the preparation of gelatin-sodium alginate / conbercept composite gel modified crosslinked amniotic membrane (Gel-SA / Conbercept@CAM) was collected and mixed, and used as the test sample solution for the experimental group; in Example 2, the rinsing solution generated by washing with sterile saline during the preparation of conbercept loaded crosslinked amniotic membrane (CAM / Conbercept) was collected and mixed, and used as the test sample solution for the control group.

[0036] In the preparation of gelatin-sodium alginate hydrogel-modified crosslinked amniotic membrane (Gel-SA@CAM) in Comparative Example 1 of Example 2, the rinsing solution generated after washing with sterile physiological saline was collected and mixed to serve as a blank control solution. The gelatin-sodium alginate hydrogel-modified crosslinked amniotic membrane (Gel-SA@CAM) does not contain conbercept, and its rinsing solution was used to subtract the background absorption of the material matrix and the rinsing solution at 280 nm, and was not included in the comparison of encapsulation efficiency results.

[0037] The experimental group, control group, and blank control solutions were filtered through a 0.22 μm filter membrane and brought to a final volume. Blank correction was performed using the blank control solution. The absorbance of the experimental group and control group samples was measured at 280 nm, and the free conbercept content in the samples was calculated based on the conbercept standard curve.

[0038] Encapsulation efficiency is calculated using the following formula: Encapsulation efficiency (%) = (M 总 -M 游离 ) / M 总 ×100%, Where: M 总In this example, M represents the total amount of Conbercept used in the experiment. 总 400 μg; M 游离 This represents the free conbercept content in the test sample solution. The free conbercept content in the test sample solution was calculated based on the conbercept standard curve. Three parallel samples were prepared for each preparation, and the final result was the average value.

[0039] Figure 3 A bar chart comparing the encapsulation efficiency of conbercept-loaded cross-linked amniotic membrane (CAM / Conbercept) and gelatin-sodium alginate / conbercept composite gel-modified cross-linked amniotic membrane (Gel-SA / Conbercept@CAM), where the horizontal axis represents different preparations and the vertical axis represents the encapsulation efficiency. Figure 3 The encapsulation efficiency of CAM / Conbercept was 26.11% ± 2.91%, while that of Gel-SA / Conbercept@CAM was 90.78% ± 3.10%. The encapsulation efficiency of the gelatin-sodium alginate / conbercept composite gel-modified cross-linked amniotic membrane (Gel-SA / Conbercept@CAM) was significantly higher than that of the conbercept-loaded cross-linked amniotic membrane (CAM / Conbercept). In the figure, "***" indicates P < 0.001, indicating a highly statistically significant difference.

[0040] The above results indicate that, compared with simply using cross-linked amnion to load conbercept, gelatin-sodium alginate hydrogel modification can significantly improve the encapsulation ability of cross-linked amnion for conbercept, which is beneficial to improving the loading and retention effect of conbercept in materials.

[0041] Example 4: Determination of swelling rate of cross-linked amniotic membrane modified with gelatin-sodium alginate hydrogel The gelatin-sodium alginate hydrogel modified crosslinked amniotic membrane (Gel-SA@CAM) prepared in Comparative Example 1 of Example 2 was used as the experimental group sample, and the gelatin-sodium alginate composite hydrogel solution (Gel-SA) prepared in Example 1 was used as the control group sample. The samples were prepared into cylindrical samples with a diameter of 15 mm and a height of 1 mm, and the swelling performance of the samples was tested by mass method.

[0042] Both the experimental and control group samples were completely immersed in deionized water at 37°C for 24 hours. After soaking, the samples were removed, and excess moisture was absorbed from the surface using filter paper. The mass of the swollen samples was measured using an analytical balance and recorded as m. The samples were then dried in a 60°C forced-air drying oven for approximately 4–6 hours until constant weight was achieved. After removal, the samples were cooled to room temperature in a desiccator and their mass was measured and recorded as m0. Three parallel samples were prepared for each preparation.

[0043] The swelling ratio is calculated using the following formula: Swelling rate (%) = (m - m0) / m0 × 100%, In the formula, m is the mass of the sample after swelling, and m0 is the mass of the sample after drying.

[0044] Figure 4 A bar chart comparing the swelling rates of gelatin-sodium alginate hydrogel-modified crosslinked amniotic membrane (Gel-SA@CAM) and gelatin-sodium alginate hydrogel (Gel-SA), where the horizontal axis represents different preparations and the vertical axis represents the swelling rate. Figure 4 The swelling ratios of Gel-SA@CAM and Gel-SA were 738.85% ± 47.31% and 800.59% ± 102.85%, respectively. The swelling ratios of gelatin-sodium alginate hydrogel-modified crosslinked amniotic membrane (Gel-SA@CAM) and gelatin-sodium alginate hydrogel (Gel-SA) were similar. In the figure, "ns" indicates P > 0.05, meaning there was no statistically significant difference between the two.

[0045] The above results indicate that modifying the cross-linked amnion with gelatin-sodium alginate hydrogel did not significantly reduce the water absorption and swelling properties of the gelatin-sodium alginate hydrogel, suggesting that the composite structure can still maintain a high water absorption capacity, which is beneficial for maintaining a local moist environment and providing a basis for subsequent drug release.

[0046] Example 5: In vitro drug release experiment 1. Establishment of the Conbercept Standard Curve Conbercept stock solution was diluted with blank medium to prepare a series of standard solutions with concentrations of 0.05 mg / mL, 0.10 mg / mL, 0.20 mg / mL, 0.30 mg / mL, 0.40 mg / mL, 0.50 mg / mL, 0.60 mg / mL, 0.70 mg / mL, and 0.80 mg / mL. The blank medium was the same as that used in the in vitro drug release experiment but without conbercept; in this example, the blank medium was sterile physiological saline. Using the blank medium as a blank control, the absorbance (A) of each standard solution was measured at a wavelength of 280 nm. A linear regression was performed with conbercept concentration (C, mg / mL) on the x-axis and absorbance (A) on the y-axis, yielding the regression equation A = 1.1689C - 0.0046, R² = 0.9955. The results showed that conbercept exhibited good linearity in the concentration range of 0.05–0.80 mg / mL, and could be used to calculate the conbercept content in the subsequent release solution.

[0047] 2. In vitro drug release experiment Four groups of samples were set up for the in vitro drug release experiment: gelatin-sodium alginate / conbercept composite gel modified cross-linked amniotic membrane group (Gel-SA / Conbercept@CAM), gelatin-sodium alginate hydrogel modified cross-linked amniotic membrane group (Gel-SA@CAM), conbercept-loaded cross-linked amniotic membrane group (CAM / Conbercept), and cross-linked amniotic membrane group (CAM). Among them, Gel-SA / Conbercept@CAM and CAM were taken from Example 1, and Gel-SA@CAM and CAM / Conbercept were taken from Example 2.

[0048] Each group of samples was placed in a centrifuge tube or culture container, with 1 mL of sterile physiological saline added to each sample as the release medium. The samples were incubated at room temperature. All release medium was collected daily and replaced with an equal volume of fresh release medium, for a total of 5 days. At each time point, an appropriate amount of the release solution was taken, filtered through a 0.22 μm filter, and the absorbance was measured at 280 nm. Using the release solutions from the Gel-SA@CAM group and the CAM group as background subtraction references to eliminate interference from gel and amniotic membrane exudates on the UV measurement, the net absorbance was substituted into the conbercept standard curve to calculate the concentration of conbercept in the release solution at each time point. The daily and cumulative release amounts were further calculated.

[0049] The results showed that the daily and cumulative release levels in the Gel-SA / Conbercept@CAM group were significantly higher than those in the CAM / Conbercept group at all time points (P < 0.01). On day 5, the cumulative release level in the Gel-SA / Conbercept@CAM group was 333.43 ± 8.68 μg, while that in the CAM / Conbercept group was 65.02 ± 10.22 μg. The Gel-SA / Conbercept@CAM group was approximately five times that of the CAM / Conbercept group, and the difference was highly statistically significant (P < 0.001).

[0050] The above results indicate that, compared to cross-linked amniotic membranes loaded with conbercept without gelatin-sodium alginate hydrogel modification, cross-linked amniotic membranes modified with gelatin-sodium alginate / conbercept composite gel significantly increase the cumulative in vitro release of conbercept, demonstrating that gelatin-sodium alginate hydrogel modification is beneficial for improving the loading and sustained release capacity of conbercept on cross-linked amniotic membranes. Therefore, cross-linked amniotic membranes modified with gelatin-sodium alginate / conbercept composite gel can serve as sustained-release drug delivery materials for conbercept on the ocular surface, providing experimental evidence for the sustained local delivery of anti-vascular endothelial growth factor drugs on the ocular surface.

[0051] Example 6: Observational Experiment and Statistical Analysis 1. Observation of the ultrastructure of the amnion using transmission electron microscopy Cross-linked amniotic membrane (CAM) and ordinary amniotic membrane (AM) were rinsed with sterile saline and then fixed in 2.5% glutaraldehyde solution. They were then rinsed with PBS buffer (pH 7.2–7.4) and fixed again with 1% osmium tetroxide. Subsequently, they underwent graded ethanol dehydration, 100% acetone impregnation, epoxy resin embedding, and ultrathin sectioning. The ultrastructure of the amniotic membrane was observed and images were captured under a transmission electron microscope. The results are shown below. Figure 1 .

[0052] Figure 1 These are transmission electron microscope (TEM) ultrastructural images of ordinary amnion and cross-linked amnion, where A represents the ordinary amnion and B represents the cross-linked amnion. Figure 1 It can be seen that the collagen fibers in ordinary amnion are relatively loosely arranged with obvious gaps between fibers; compared with ordinary amnion, the collagen fibers in cross-linked amnion are more tightly arranged with a significantly increased fiber density.

[0053] The above results indicate that crosslinking treatment can enhance the compactness of the amniotic matrix structure and improve the stability of the amniotic structure.

[0054] 2. Observation of hydrogel morphology The appearance of gelatin-sodium alginate hydrogel (Gel-SA) and gelatin-sodium alginate hydrogel-modified crosslinked amniotic membrane (Gel-SA@CAM) was observed by the naked eye and photographed. The results are shown in the figure. Figure 2 .

[0055] Figure 2 The images show a comparison of the appearance of Gel-SA hydrogel and Gel-SA@CAM composite gel, where C represents Gel-SA hydrogel and D represents Gel-SA@CAM composite gel. Figure 2 It can be seen that Gel-SA is a colorless and transparent homogeneous gel with a uniform overall appearance and good moldability; Gel-SA@CAM is a colorless and semi-transparent hydrogel with the amnion evenly wrapped around the bottom of the gel, and the overall structure is intact, with no obvious cracks, detachment or delamination.

[0056] The above results indicate that gelatin-sodium alginate hydrogel can bind well with cross-linked amniotic membrane to form a hydrogel-modified cross-linked amniotic membrane composite material with intact appearance and stable structure.

[0057] 3. Statistical Analysis Statistical analysis was performed using R language 4.3.1. Figure 5 The graph shows the results of an in vitro drug release experiment. A is the daily release curve, B is the cumulative release curve, and C is a bar chart comparing the cumulative release on day 5.

[0058] Depend on Figure 5A indicates that the Gel-SA / Conbercept@CAM group consistently released conbercept from day 1 to day 5, and the daily release at each time point was higher than that of the CAM / Conbercept group; Figure 5 As shown in Figure B, the cumulative release amount in the Gel-SA / Conbercept@CAM group continuously increased with prolonged release time and was significantly higher than that in the CAM / Conbercept group, indicating that the gelatin-sodium alginate / conbercept composite gel-modified cross-linked amniotic membrane has a higher conbercept release capacity; Figure 5 As shown in Figure C, on day 5, the cumulative release of the Gel-SA / Conbercept@CAM group was 333.43±8.68 μg, while the cumulative release of the CAM / Conbercept group was 65.02±10.22 μg. The Gel-SA / Conbercept@CAM group was approximately 5 times that of the CAM / Conbercept group. "***" in the figure indicates P < 0.001, meaning the difference between the two groups was highly statistically significant.

[0059] The above results indicate that, compared with the cross-linked amniotic membrane loaded with conbercept without gelatin-sodium alginate hydrogel modification, the cross-linked amniotic membrane modified with gelatin-sodium alginate / conbercept composite gel can significantly increase the cumulative release of conbercept, indicating that gelatin-sodium alginate hydrogel modification is beneficial to improving the loading and sustained release capacity of conbercept on the cross-linked amniotic membrane.

[0060] Example 7: In vitro collagenase degradation experiment of ordinary amniotic membrane and cross-linked amniotic membrane To evaluate the effect of UV / riboflavin cross-linking treatment on the anti-enzymatic degradation ability of amniotic membrane, ordinary amniotic membrane (AM) and cross-linked amniotic membrane (CAM) were subjected to in vitro collagenase degradation experiments.

[0061] Ordinary amniotic membrane (AM) and cross-linked amniotic membrane (CAM) were cut into 1cm × 1cm pieces, with 5 pieces per group at each detection time point. Each piece was dried in a 60℃ forced-air drying oven until constant weight, and the initial dry weight was recorded as W0. A PBS solution containing 0.25% type I collagenase was prepared as the degradation solution, with a pH of 7.4. Each amniotic membrane piece was completely immersed in 1 mL of the degradation solution and incubated in a 37℃ constant-temperature shaker at 80 rpm. During incubation, the corresponding samples were removed at each detection time point, and the degradation solution was replaced for samples continuing incubation.

[0062] The degradation treatment of the corresponding samples was terminated on days 1, 3, 5, and 7 of incubation. After the degradation was terminated, the entire reaction system was centrifuged at 12,000 rpm for 15 min. The supernatant containing degraded collagen fragments was discarded, and the remaining solid residue was collected and dried again in a 60℃ forced-air drying oven until constant weight. The remaining dry weight was then weighed and recorded as W. t .

[0063] The degradation rate is calculated using the following formula: Degradation rate (%) = (W0 - W) t ) / W0×100%.

[0064] In the formula, W0 is the initial dry weight of the amnion before degradation, W t The remaining dry weight of the amnion after collagenase degradation treatment.

[0065] The experimental results are shown in Figure 6 . Figure 6 This graph shows the changes in in vitro collagenase degradation rates of ordinary amnion (AM) and cross-linked amnion (CAM), where the horizontal axis represents incubation time and the vertical axis represents degradation rate. Figure 6 It was found that ordinary amnion (AM) was completely degraded on day 1 of incubation, with a degradation rate of 100%, and no obvious solid residue was observed after centrifugation. The remaining dry weight could not be measured at subsequent time points. Cross-linked amnion (CAM) degraded more slowly in the collagenase degradation solution, and the degradation rate gradually increased with prolonged incubation time. Specifically, the degradation rate of CAM was 8.25% ± 1.34% on day 1, 18.67% ± 2.41% on day 3, 32.14% ± 4.02% on day 5, and 49.35% ± 5.87% on day 7. The differences in degradation rate at each time point were statistically significant (P < 0.01).

[0066] The above results indicate that, compared with ordinary amniotic membrane (AM), cross-linked amniotic membrane (CAM) has a stronger resistance to collagenase degradation, suggesting that UV / riboflavin cross-linking treatment can improve the structural stability of the amniotic membrane matrix and delay the degradation process of the amniotic membrane in the collagenase environment, thereby helping to prolong the retention time of the amniotic membrane material in the ocular surface environment.

Claims

1. A gelatin-sodium alginate / conbercept composite gel-modified cross-linked amnion, characterized in that: It includes a cross-linked amnion and a gelatin-sodium alginate / conbercept composite gel layer bonded to the surface of the cross-linked amnion; the gelatin-sodium alginate / conbercept composite gel layer is formed by calcium ion cross-linking of a gelatin-sodium alginate composite hydrogel solution containing conbercept and covers the surface of the cross-linked amnion.

2. The gelatin-sodium alginate / conbercept composite gel modified crosslinked amnion according to claim 1, characterized in that: Cross-linked amnion is amnion that has been cross-linked with riboflavin and ultraviolet light.

3. The gelatin-sodium alginate / combercept composite gel modified crosslinked amnion according to claim 2, characterized in that: Cross-linked amniotic membrane is obtained by incubating amniotic membrane with riboflavin solution and then cross-linking it with ultraviolet light.

4. The gelatin-sodium alginate / conbercept composite gel modified crosslinked amnion according to claim 1, characterized in that: The gelatin-sodium alginate composite hydrogel solution is prepared by dissolving 4% (w / v) gelatin and 2% (w / v) sodium alginate in deionized water.

5. The gelatin-sodium alginate / combercept composite gel modified crosslinked amnion according to claim 1, characterized in that: The gelatin-sodium alginate / combercept composite gel layer is formed by cross-linking with calcium chloride solution.

6. The gelatin-sodium alginate / conbercept composite gel modified crosslinked amnion according to claim 1, characterized in that: The cross-linked amniotic membrane was a circular amniotic membrane sheet with a diameter of 15 mm; when preparing each gelatin-sodium alginate / conbercept composite gel modified cross-linked amniotic membrane sheet, the dosage of conbercept was 400 μg.

7. A method for preparing cross-linked amniotic membrane modified with gelatin-sodium alginate / conbercept composite gel, characterized in that: Includes the following steps: S1. The amnion is pretreated and then cross-linked with riboflavin and ultraviolet light to obtain a cross-linked amnion. S2. Add gelatin and sodium alginate to deionized water, heat and stir to dissolve, and obtain gelatin-sodium alginate composite hydrogel solution. S3. Add conbercept to the gelatin-sodium alginate composite hydrogel solution and mix well to obtain gelatin-sodium alginate / conbercept composite gel. S4. The gelatin-sodium alginate / conbercept composite gel was loaded onto the surface of the cross-linked amnion and subjected to calcium ion cross-linking treatment to obtain the gelatin-sodium alginate / conbercept composite gel modified cross-linked amnion.

8. The method for preparing cross-linked amnion modified with gelatin-sodium alginate / combercept composite gel according to claim 7, characterized in that: In S1, the pretreated amnion was completely immersed in a 0.1% riboflavin solution and incubated for 10 minutes. Then, it was subjected to crosslinking treatment by vertical irradiation with 365nm ultraviolet light for 4 minutes. The ultraviolet light irradiation intensity was 30mW / cm², and the total irradiation energy was 7.2J / cm².

9. The method for preparing cross-linked amnion modified with gelatin-sodium alginate / combercept composite gel according to claim 7, characterized in that: In S4, gelatin-sodium alginate / conbercept composite gel was dropped onto the surface of the cross-linked amnion and incubated at 4°C for 30 min, followed by cross-linking at 4°C for 1 min using 3% (w / v) calcium chloride solution.

10. Application of gelatin-sodium alginate / conbercept composite gel modified cross-linked amniotic membrane in the preparation of conbercept ocular sustained-release drug delivery materials.

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