New application of cross-linked decellularized bovine amniotic membrane and manganese carbon dot combined use, compound for repairing alkali burn cornea and preparation method of compound

Abstract

The invention provides an application of a cross-linked decellularized bovine amniotic membrane (CAM) and manganese carbon dots (Mn CDs) combined in preparation of an ophthalmic medical material for alkali burn cornea repair. The invention further provides a compound for repairing the alkali burn cornea. Cell and animal experiments show that the compound CDs (at) CAM has good biocompatibility and strong antioxidant and anti-inflammatory effects, can significantly promote corneal epithelium regeneration and inhibit corneal neovascularization and scar generation, and provides a new strategy for treatment of alkali burn cornea and various corneal epithelium injuries and diseases.

Description

Technical Field

[0001] The invention relates to the use of cross-linked decellularized amniotic membrane (CAM) and manganese carbon dots (Mn CDs) in preparing ophthalmic medical materials for repairing alkali-burned cornea, and a composite for repairing alkali-burned cornea and a preparation method thereof. Background Art

[0002] The cornea is a transparent tissue structure in the front of the eyeball and is prone to damage. Corneal damage is often accompanied by the secretion of irregular collagen matrix, which leads to light scattering and can cause blindness in severe cases. Epidemiological surveys show that more than 10 million patients worldwide are blinded by corneal diseases or injuries each year. Although corneal transplantation technology has made significant progress, the severe shortage of donor corneas limits its clinical application. In this context, it is necessary to study alternative materials for corneal treatment. Amniotic membrane (AM), a biomaterial with unique biological properties, has shown broad application prospects in the treatment of corneal diseases. Amniotic membrane has unique biological properties: its matrix components can provide a suitable microenvironment for cell growth. Studies have shown that the protein-polysaccharide complexes such as type I and type III collagen fibers and glycosaminoglycans contained in amniotic membrane can promote epithelial cell proliferation and differentiation, enhance cell adhesion, and thus accelerate the repair of corneal epithelial damage. In addition, amniotic membrane can also inhibit the excessive proliferation of corneal fibroblasts mediated by inflammatory cells. These characteristics make amniotic membrane show significant advantages in promoting wound healing, reducing inflammatory response and inhibiting scar formation, making it an ideal biomaterial for corneal repair. At present, human amniotic membrane (HAM) is mainly used for clinical treatment, but its source is limited and involves medical ethical issues. Therefore, it is of great clinical significance to explore biomaterials that can replace human amniotic membrane. Animal-derived amniotic membrane may be a potential alternative because of its wide source, but its immunogenicity needs to be addressed. Studies have shown that decellularization can effectively remove cellular components in tissues and significantly reduce the immunogenicity of materials. Decellularized amniotic membrane (DAM) not only retains the original bioactive components of amniotic membrane such as collagen and glycosaminoglycans, but also has extremely low immunogenicity because it removes cellular components. However, decellularized amniotic membrane still has shortcomings such as low light transmittance, poor mechanical properties and poor resistance to enzymatic hydrolysis. Existing studies have confirmed that cross-linking treatment can enhance the connection between fibers, thereby improving the mechanical properties and anti-enzymatic properties of the material [F. Raiskup, E. Spoerl, Corneal crosslinking with riboflavin and ultraviolet AI Principles, Ocul. Surf. 11 (2) (2013) 65-74.].

[0003] The occurrence of corneal injury is closely related to oxidative stress and inflammatory response. Chemical eye injury (CEI) accounts for 10%-22% of ocular trauma, and its incidence is second only to intraocular foreign bodies (43.42%), ranking second among occupational eye injuries (12.68%). The key to the treatment of chemical eye injury is to timely control the oxidative stress and inflammatory response of the eye, which directly affect the degree of injury and prognosis. Among them, alkali burns account for about 60% of chemical eye injuries, because they can undergo saponification reaction with cell membranes, leading to the production of soluble substances, thereby causing persistent damage to the cornea and intraocular tissues, and the degree of damage is more serious than acid burns. Studies have shown that in the process of corneal alkali burns, oxidative stress occurs earlier than inflammatory response, and continuous high levels of oxidative stress and excessive inflammatory response can cause complications such as large-area corneal defects, scar formation, and corneal neovascularization (CNV). Therefore, if amniotic membrane transplantation can play its traditional scaffold role and provide a matrix environment for cell growth while also having the dual functions of antioxidant and anti-inflammatory, it will be expected to significantly improve the treatment effect of alkali-burned cornea.

[0004] Antioxidant nanozymes are a class of nanomaterials with enzyme-like catalytic activity. With their advantages such as versatility, multi-enzyme activity and high stability, they have been widely used in the biomedical field. With the continuous growth of demand for enzyme-catalyzed biotherapy, the research on artificial enzymes (especially metalloenzymes) has made significant progress. At present, researchers have successfully synthesized and systematically studied a variety of nanozyme materials including carbon-based materials, metals and their oxides, which can simulate the biological functions of natural enzymes such as catalase (CAT) and superoxide dismutase (SOD). Experiments have confirmed that these nanozyme materials exhibit excellent reactive oxygen species (ROS) scavenging capabilities both in vitro and in vivo. Among the many nanozyme materials, transition metal-doped carbon dots have attracted much attention due to their variable valence states and diverse surface functional groups at the metal center. The introduction of metal ions not only enhances the surface functional properties of carbon dots, but also promotes electron transfer during the reaction process, making them exhibit excellent multi-enzyme catalytic activity. This multi-enzyme activity is of great value in regulating oxidative stress in pathological tissues and can effectively alleviate excessive oxidative stress caused by the imbalance between antioxidants and pro-oxidants in tissues. Studies have shown that reducing the level of oxidative stress can significantly inhibit tissue inflammatory response, thereby reducing the occurrence of related complications.

[0005] There are no reports on the combined use of manganese carbon dots (Mn CDs) and amniotic membrane. Summary of the invention

[0006] Based on the above findings, if transition metal-doped carbon dots with multi-enzyme activity are combined with amniotic membrane, it is expected to provide an innovative strategy for the treatment of alkali-burned cornea. This composite therapy can not only provide a suitable growth microenvironment and bioactive components for wound healing, but also enhance the therapeutic effect by effectively removing reactive oxygen and reducing inflammatory response - promoting corneal epithelial regeneration, inhibiting scar formation and reducing corneal neovascularization.

[0007] The invention provides use of cross-linked decellularized amniotic membrane (CAM) and manganese carbon dots (Mn CDs) in combination in preparing an ophthalmic medical material for repairing alkali-burned cornea.

[0008] Among them, the mass ratio of cross-linked decellularized bovine amniotic membrane (CAM) and manganese carbon dots (Mn CDs) is:

[0009] Cross-linked decellularized bovine amniotic membrane (CAM): manganese carbon dots (Mn CDs) = 374-1499:1.

[0010] Preferably, the mass ratio of cross-linked decellularized amniotic membrane (CAM) to manganese carbon dots (Mn CDs) is:

[0011] Cross-linked decellularized bovine amniotic membrane (CAM): manganese carbon dots (Mn CDs) = 599:1.

[0012] The ophthalmic medical material promotes corneal epithelial regeneration and inhibits neovascularization and scar formation.

[0013] The present invention also provides a composite for repairing alkali-burned cornea, which is composed of the following raw materials in weight ratio:

[0014] Cross-linked decellularized bovine amniotic membrane (CAM) 374-1499 parts, manganese carbon dots (Mn CDs) 1 part.

[0015] The present invention provides a composite material for repairing alkali-burned cornea, which is composited from the following raw materials in weight ratio:

[0016] 599 parts of cross-linked decellularized bovine amniotic membrane (CAM) and 1 part of manganese carbon dots (Mn CDs).

[0017] Wherein, the manganese carbon dots (Mn CDs) are prepared by solvent thermal synthesis, and the specific preparation method is:

[0018] Manganese dichloride (MnCl 2 ) and glutathione (GSH) are completely dissolved in formamide; the mixture is then placed in a polytetrafluoroethylene-lined hot liquid autoclave to react at high temperature and high pressure to generate manganese carbon dots (Mn CDs); the manganese dichloride (MnCl 2The mass ratio of ) to glutathione (GSH) was 1:6.35; the reaction temperature was 180°C and the reaction time was 4h;

[0019] After the reaction, the solution was cooled to room temperature for filtration and dialyzation, and then concentrated using a rotary evaporator to prepare Mn CDs.

[0020] The present invention also provides a method for preparing the composite for repairing alkali-burned cornea, which comprises the following steps:

[0021] a. Weigh the raw materials of each weight ratio;

[0022] b. The cross-linked decellularized amniotic membrane (CAM) was immersed in a manganese carbon dots (Mn CDs) solution and placed on an oscillator running at 100 rpm for 48 hours.

[0023] The invention provides use of the composite in preparing a medical material for ophthalmic diseases used for repairing alkali-burned cornea.

[0024] Corneal alkali burns are a common ocular emergency that can lead to blindness and are characterized by inflammation and delayed epithelial healing due to elevated oxidative stress in the intraocular environment. Reducing reactive oxygen species (ROS) levels and inflammatory responses are critical for the development of corneal repair materials. Amniotic membrane (AM) is commonly used for ocular surface repair, but faces limitations such as limited sources and rapid degradation. The present invention develops a cross-linked decellularized bovine amniotic membrane (CAM) with excellent light transmittance, mechanical strength, and enzyme resistance. By combining it with manganese carbon dots (Mn CDs), the resulting composite material (CDs@CAM) retains the excellent physical properties of CAM while introducing the multi-enzyme activity of Mn CDs. Characterization of various materials showed that CDs@CAM has excellent CAT enzyme activity, SOD enzyme activity, and scavenging ability for hydroxyl radicals and nitrogen radicals. In addition, cell and animal experiments also showed that CDs@CAM has good biocompatibility, strong antioxidant and anti-inflammatory effects, can significantly promote corneal epithelial regeneration, inhibit neovascularization and scar formation, and provides a new strategy for the treatment of alkali-burned cornea and various corneal epithelial injuries and diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Preparation and properties of amniotic membrane scaffolds (A) Schematic diagram of the process of preparing DAM and CAM using FAM (B) Sirius red staining (C) Hematoxylin-eosin (H&E) staining (D) Scanning electron microscopy images (E) Transmittance curve and transparency (F) Stress-strain curve (G) Tensile strength (H) Fourier transform infrared spectroscopy (FTIR) analysis (I) Thermogravimetric analysis (TGA) (J) In vitro enzyme degradation curves Data are expressed as Mean ±

[0026] SD (n=3), **P<0.01, ***P<0.001, and ****P<0.0001;

[0027] Figure 2 .Periodic acid Schiff (PAS) staining of FAM, DAM and CAM;

[0028] Figure 3 . Transparency of FAM, DAM and CAM;

[0029] Figure 4 .Thickness of FAM, DAM and CAM;

[0030] Figure 5 .Water content of FAM, DAM and CAM;

[0031] Figure 6 .Elongation at break, maximum load and elastic modulus of FAM, DAM and CAM;

[0032] Figure 7 Preparation and biomimetic enzyme activity of CDs@CAM (A) Schematic diagram of the preparation process of Mn CDs and CDs@CAM (B) SEM elemental mapping analysis of CAM and CDs@CAM (C) FTIR analysis of CDs and CDs@CAM (D) Transmittance (E) Thermogravimetric analysis (TGA) curves of CAM and CDs@CAM (F) In vitro enzymatic degradation study (G) Schematic diagram of multienzyme activity of CDs@CAM (H, I) Determination of hydroxyl radical (·OH) scavenging ability (HRSA) of CDs@CAM using UV-visible spectrometer and electron spin resonance (ESR) assay (J, K) Determination of SOD-like enzyme activity of CDs@CAM using UV-visible spectrometer and ESR assay (L) CAT-like enzyme activity of CDs@CAM (M) Reactive nitrogen (RNS) scavenging ability of CDs@CAM Data are expressed as Mean±SD (n=3), *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001; ns, no significant difference;

[0033] Figure 8 . Transmission electron microscopy (TEM) results, particle size distribution analysis results and X-ray photoelectron spectroscopy (XPS) full spectrum analysis of Mn CDs;

[0034] Fig. 9 .Cytocompatibility of CDs@CAM;

[0035] Fig.10.In vitro antioxidant and anti-inflammatory properties of CDs@CAM (A) HCECs cell fluorescence image results using 2′,7′-dichlorofluorescein diacetate as a probe (B) ROS expression levels of HCECs in fluorescence staining images of different groups (C) Flow cytometry results of RAW264.7 cells in different groups (D) Immunofluorescence images of TNF-α, IL-1β and IL-6 immunofluorescence staining of RAW264.7 cells in different groups (EG) ELISA results of TNF-α, IL-1β and IL-6 in RAW264.7 cells in different groups Data are expressed as Mean±SD (n=3); *p<0.05, **p<0.01 and ****p<0.0001; ns, no significant difference;

[0036] Fig.11 CDs@CAM treatment of alkali-burned cornea promotes corneal epithelialization and thickness recovery (A) Schematic diagram of the treatment timeline and postoperative examination method of animal experiments on alkali-burned cornea (B) Representative slit lamp photos and sodium fluorescein staining images of alkali-burned corneas in different groups (C) Representative anterior segment optical coherence tomography (AS-OCT) images taken on days 1, 3, 7, and 28 (D) Corneal epithelialization rate of alkali-burned corneas in different groups (E) Comparison of corneal thickness from AS-OCT images of normal group, control group, CAM treatment group and CDs@CAM treatment group Data are expressed as Mean±SD (n=3), ***p<0.001 and ****p<0.0001; ns, no significant difference;

[0037] Fig.12 .H&E staining of SD rat cornea on day 7;

[0038] Fig.13 .Histological evaluation of the anti-inflammatory efficacy of CDs@CAM on day 7 (A) Immunofluorescence images of TNF-α, IL-1β, and IL-6 in corneal tissues of different groups (BtoD) Quantitative analysis of the expression of the three inflammatory cytokines in the immunofluorescence staining images of corneal tissues Data are expressed as Mean±SD (n=3), *p<0.05 and ****p<0.0001;

[0039] Fig.14.Histological evaluation of the corneal repair effect of CDs@CAM on day 28 (A) H&E staining images of corneas in different groups on day 7 (B) Analysis of corneal epithelial thickness in different groups in H&E images (C) Immunofluorescence staining of corneal function index (ZO-1, CD31 and α-SMA) in corneal tissue sections of SD rats (D to F) Quantitative analysis of the expression of ZO-1, CD31 and α-SMA in corneal immunofluorescence staining images Data are expressed as Mean±SD (n=3), *p<0.05, ***p<0.001, and ****p<0.0001. DETAILED DESCRIPTION

[0040] The present invention first uses fresh bovine amniotic membrane (FAM) to prepare decellularized bovine amniotic membrane (DAM). Specifically, decellularized bovine amniotic membrane is obtained by removing the immunogenic components in fresh bovine amniotic membrane, and then crosslinked decellularized bovine amniotic membrane (CAM) with excellent light transmittance, thermal stability, mechanical properties and resistance to enzymatic degradation is prepared by glutaraldehyde crosslinking method (Crosslinked decellularized bovine amniotic membrane can be prepared with reference to the following literature):

[0041] [1]APLynch,M.Ahearne,Strategies for developing decellularizedcorneal scaffolds.Exp Eye Res.108(2013)42-7.

[0042] [2]JYLai,DHMa,Glutaraldehyde cross-linking of amniotic membranesaffects their nanofibrous structures and limbal epithelial cell culturecharacteristics,Int.J.Nanomedicine.8(2013)4157-68.

[0043] Then, the cross-linked decellularized amniotic membrane (CAM) was immersed in a manganese carbon dots (MnCDs) solution to finally prepare a composite material of cross-linked decellularized amniotic membrane and manganese carbon dots (CDs@CAM).

[0044] The experimental results show that CDs@CAM not only has the high transmittance, good thermal stability, excellent mechanical properties and resistance to enzymatic degradation of CAM, but also exhibits the multi-enzyme activity of manganese carbon dots, including catalase (CAT-like) activity, superoxide dismutase (SOD-like) activity and the ability to scavenge hydroxyl radicals and nitrogen radicals. In in vitro cell experiments, CDs@CAM exhibited significant antioxidant and anti-inflammatory effects. More importantly, the experimental results of the corneal alkali burn animal model showed that the material showed significant advantages in exerting anti-inflammatory effects and promoting corneal epithelial healing, indicating that it has certain application potential in corneal repair treatment.

[0045] Example 1 Preparation of the composite for repairing alkali-burned cornea of ​​the present invention

[0046] 1. Materials and Methods

[0047] 1.1 Preparation of cross-linked decellularized bovine amniotic membrane (CAM)

[0048] First, fresh bovine amniotic membrane (FAM) was obtained by stripping from placental tissue. After strict sterilization, it was pre-treated by defatting, desugaring, etc., and then frozen. In order to obtain decellularized bovine amniotic membrane (DAM), physical and chemical methods were used in sequence: first, the cell structure was destroyed by repeated freezing and thawing, then the cell membrane components were removed by sodium deoxycholate solution, and finally the nucleic acid residues were digested by deoxyribonuclease and ribonuclease solution. On this basis, in order to further improve the material properties, glutaraldehyde was used as a cross-linking agent to cross-link DAM, and cross-linked decellularized bovine amniotic membrane (CAM) was successfully prepared.

[0049] 1.2. Physical and biological properties of cross-linked acellular bovine amniotic membrane (CAM)

[0050] The three amniotic membrane materials (including FAM, DAM and CAM) were fixed in 4% (v / v) formalin solution, embedded in paraffin and sliced, and stained with hematoxylin-eosin (H&E), periodic acid-Schiff (PAS) and Sirius red, respectively. The transparency of the samples was evaluated by a digital camera, and the transmittance in the wavelength range of 400-800nm ​​was measured using a UV-visible spectrophotometer. The thickness of the material was accurately measured using a micrometer; the water content was determined by weighing, that is, the dried sample (W 0 ) were immersed in phosphate buffered saline (PBS) at 37°C for 12 hours and then weighed (W t), according to the formula: Water content (%) = [(W t -W 0 ) / W t ]×100% to calculate the water content. The mechanical properties were evaluated using a universal testing machine under the following test conditions: load capacity 10N, tensile rate 10mm / min, and initial clamp spacing 10mm. The chemical properties of the materials were analyzed using thermogravimetric analysis (TGA) and Fourier transform infrared spectroscopy (FTIR). The TGA test parameters were: nitrogen flow rate 25mL / min, heating rate 20℃ / min, temperature range from room temperature to 600℃, and sample mass 3-5mg. The in vitro degradation experiment used a circular sample with a diameter of 10mm (n≥3) and an initial weight of W. 0 Incubate in PBS solution containing 50 U / mL collagenase at 37°C, take samples regularly, wash, dry and weigh (W t ), according to the formula: Residual mass ratio (%) = [(W 0 -W t ) / W 0 ]×100% to calculate the degradation rate. All experiments used the same parameters to ensure the comparability and reliability of the results.

[0051] 1.3. Preparation of manganese carbon dots (Mn CDs)

[0052] Mn CDs were prepared by solvothermal synthesis. 2 ) 0.0378g and glutathione (GSH) 0.24g were completely dissolved in 60mL formamide. The mixture was then placed in a polytetrafluoroethylene-lined hydrothermal autoclave for high temperature and high pressure reaction to produce Mn CDs. After the reaction was completed, the solution was cooled to room temperature for filtration and dialysis, and then concentrated using a rotary evaporator to prepare Mn CDs for further analysis.

[0053] 1.4. Preparation of Mn-Cdots and Cross-linked Decellularized Bovine Amniotic Membrane Composite (CDs@CAM)

[0054] CAM360mg was immersed in manganese carbon dots (MnCDs) solutions with concentrations of 24μg / mL, 60μg / mL, and 96μg / mL, respectively, to prepare CDs@CAM composite materials with three concentrations.

[0055] The excess water on the CAM surface was first wiped off with filter paper, and then it was immersed in different concentrations of manganese carbon dots (Mn CDs) solutions and placed on an oscillator running at 100 rpm for 48 h.

[0056] The beneficial effects of the present invention are demonstrated by specific efficacy tests as follows.

[0057] Example 1 Efficacy test of the compound of the present invention

[0058] 1. Experimental methods

[0059] 1.1. Reactive oxygen species (ROS) scavenging ability of Mn-C dots and cross-linked decellularized bovine amniotic membrane (CDs@CAM) composite

[0060] The present invention uses a variety of methods to detect the free radical scavenging ability of the sample. First, 5,5'-tetramethylbenzidine (TMB) is used as a probe to detect the hydroxyl radical (·OH) scavenging ability by UV-visible spectrophotometer. The specific method is: FeSO 4 (4mM) and H 2 O 2 (200mM) was reacted for 5min to generate ·OH, and then TMB (2mM) was added to form a blue solution. After a circular sample with a diameter of 10mm was added to the solution, the fading phenomenon of the solution was observed, and the change of the characteristic absorption peak at 652nm was detected by UV-visible spectrophotometer. In addition, the ·OH scavenging ability was detected by electron spin resonance (ESR) spectroscopy. This method generates ·OH through a Fenton-like reaction, adds the test sample and DMPO (100mM), reacts for 2min, and records the ESR spectrum.

[0061] For superoxide anion (O 2 The present invention adopts two methods to evaluate the scavenging ability of superoxide dismutase (SOD): firstly, a superoxide dismutase (SOD) activity detection kit is used to generate SOD through the reaction of xanthine oxidase and xanthine. 2 ·-, nitro blue tetrazolium (NBT) was reduced to formazan with characteristic absorption at 560nm. A 10mm circular sample was mixed with NBT, enzyme working solution and reaction initiator, incubated at 37℃ for 30min, and the absorbance at 560nm was measured using a UV-visible spectrophotometer to evaluate the SOD-like activity. At the same time, the ESR method was used to detect the O generated by the sample in the methanol UV irradiation system. 2 ·-'s scavenging ability, the ESR spectra were recorded 5 min after adding samples and DMPO (100 mM).

[0062] CAT-like activity was detected by using hydrogen peroxide (H 2 O 2 ) content determination kit. This method is based on H 2 O 2 With titanium sulfate (Ti(SO 4 ) 2 ) forms a yellow titanium peroxide complex with a characteristic absorption peak at 415nm. 2 O 2(2mM) After reacting for 5 minutes, the kit working solutions II and III were added in sequence. After reacting for 5 minutes, the supernatant was removed by centrifugation, and the precipitate was dissolved with working solution IV. Finally, the characteristic absorption peak at 415 nm was measured using a UV-visible spectrophotometer to calculate the catalase-like activity.

[0063] 1.2. Ability of Mn-C dots and cross-linked decellularized bovine amniotic membrane composite (CDs@CAM) to scavenge nitrogen radicals

[0064] 2,2-Diphenyl-1-picrylhydrazyl (DPPH) can be used to determine the reactive nitrogen (RNS) scavenging activity of antioxidants. Each group of materials was added to an ethanol solution containing DPPH (1 mg / mL), placed at room temperature for 30 minutes, and the absorbance at 517 nm was measured using a UV-visible spectrophotometer.

[0065] 1.3. Biocompatibility of Mn-Cdots and Cross-linked Decellularized Bovine Amniotic Membrane Composites (CDs@CAM)

[0066] The cytocompatibility of CDs@CAM was evaluated by live / dead cell staining and CCK-8 assay. Human corneal epithelial cells (HCECs) were cultured at 5 × 10 4 The cells were seeded at a density of 100 μl / well in 24-well plates containing CAM and CDs@CAM and cultured for 1, 3, and 5 days, respectively. Subsequently, the cells were stained with FDA / PI and photographed using confocal laser scanning microscopy (CLSM) to determine cell viability. For the CCK-8 assay, 100 μl of CCK-8 reagent was added to each well and incubated for 1 to 2 hours. After incubation, the solution in each well was carefully transferred to a 96-well plate and the absorbance was measured at 450 nm using a microplate reader.

[0067] 1.4. Antioxidant and anti-inflammatory activities of Mn-C dots and cross-linked decellularized bovine amniotic membrane composite (CDs@CAM)

[0068] To evaluate the antioxidant and anti-inflammatory activities of CDs@CAM, the present invention conducted a series of cell experiments. First, the DCFH-DA fluorescent probe was used to detect the ROS scavenging ability of CDs@CAM in human corneal epithelial cells (HCECs). The specific method was as follows: HCECs were incubated at 5×10 4 The cells were seeded at a density of 100 μg / well in a 24-well plate and cultured for 12 h. 2 O 2 The cells were stimulated for 2 h, and samples from different treatment groups were added and incubated for another 24 h. Finally, the fluorescence intensity was observed by confocal laser scanning microscopy (CLSM).

[0069] The anti-inflammatory activity was evaluated by immunofluorescence staining, flow cytometry, and ELISA. In the immunofluorescence experiment, RAW 264.7 cells were plated at 5×10 4 The cells were inoculated at a density of 10 cells / well in a 24-well plate and cultured for 12 hours, then stimulated with 1 μg / ml LPS for 12 hours. After adding samples from each group and incubating for 24 hours, immunofluorescence staining of three inflammatory factors, IL-1β, IL-6, and TNF-α, was performed, and fluorescence images were obtained using CLSM. In flow cytometry and ELISA experiments, RAW 264.7 cells were inoculated at 4×10 5 Cells were inoculated at a density of 100 cells / well in a 6-well plate and cultured for 12 hours. After LPS stimulation for 12 hours, the cell suspension and culture supernatant were collected. The cell suspension was used to analyze the cell phenotype by flow cytometry, and the culture supernatant was used to detect the secretion levels of IL-1β, IL-6 and TNF-α using ELISA kits. Cells that were not stimulated or treated in any way were used as negative controls in the experiment. All fluorescence images were analyzed for fluorescence intensity using Image J software.

[0070] 1.5. Manganese carbon dots and cross-linked decellularized bovine amniotic membrane composite (CDs@CAM) for the treatment of alkali-burned cornea

[0071] All in vivo studies were fully evaluated and authorized by the Animal Care and Use Committee of Sichuan University (KS2023352) and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Research Council of the United States. Six-week-old male SD rats weighing between 180 and 220 g and with healthy corneas were obtained from Chengdu Dashuo Laboratory Animal Co., Ltd. and housed at the West China Animal Management Center of Sichuan University for 7 days to adapt to the environment (ambient temperature of 23 °C, relative humidity of 50%, and 12-hour light / dark cycle). A filter paper disc with a diameter of 3 mm was immersed in a 0.5 mM NaOH solution. It was then removed and placed in the center of the cornea of ​​the SD rat for 90 seconds to produce corneal alkali burns. After removing the filter paper, the cornea was rinsed with saline for 60 seconds. The rats with successfully established alkali burn models were randomly divided into 4 experimental groups: normal group, control group (modeling without any intervention), CAM group and CDs@CAM group (the dosage of CAM group and CDs@CAM group was 6 mg, among which, the dosage of CDs@CAM group was 60 μg / mL), and the normal rat cornea without modeling was used as negative control. Regular follow-up was performed by slit lamp and continuous anterior segment optical coherence tomography (AS-OCT) examinations to observe corneal transparency and thickness, and Image J software was used to calculate the epithelial healing rate and corneal thickness recovery.

[0072] Histological evaluation

[0073] In order to evaluate the therapeutic effect of CDs@CAM in vivo, the present invention established a corneal alkali burn model in SD rats. On the 7th and 28th days after surgery, the experimental animals were killed by cervical dislocation, and the eyeballs were removed and fixed with 4% paraformaldehyde, embedded in paraffin and sliced. The slices were processed as follows: samples at all time points were stained with hematoxylin-eosin (H&E); samples 7 days after surgery were immunofluorescently stained for three inflammatory factors, IL-1β, IL-6 and TNF-α; samples 28 days after surgery were immunofluorescently stained for corneal repair-related markers. Image J software was used to quantitatively analyze the staining results, including corneal epithelial thickness measurement and fluorescence intensity calculation, to obtain objective quantitative evaluation data.

[0074] Data Analysis

[0075] All data are expressed as mean ± SD, and statistical analyses were performed using GraphPad Prism (version 10.3.1) and Origin (version 9.8.0.20) software.

[0076] 2. Results and Discussion

[0077] 2.1. Preparation and characterization of cross-linked acellular bovine amniotic membrane (CAM)

[0078] like Figure 1 As shown in A, fresh bovine amniotic membrane (FAM) was repeatedly frozen and thawed, and then treated with sodium deoxycholate, deoxyribonuclease and ribonuclease solutions in sequence to obtain decellularized bovine amniotic membrane (DAM). After DAM was immersed in a glycerol aqueous solution, glutaraldehyde (GTA) was added for cross-linking reaction, and CAM was finally obtained. H&E staining result analysis ( Figure 1 C) showed that there was a complete cell structure in FAM, while the cell components in DAM were completely removed, confirming that the decellularization process was successful. It is worth noting that the internal structure of the amniotic membrane was loose after decellularization, and after cross-linking, the connection between collagen fibers was significantly enhanced. This phenomenon is attributed to the formation of a large number of covalent bonds between collagen molecules during the cross-linking process, which led to an increase in the degree of molecular aggregation. Further histochemical staining results confirmed that decellularization and cross-linking treatment did not significantly affect the basic composition of the amniotic membrane. Sirius red staining ( Figure 1 B) and periodic acid Schiff (PAS) staining ( Figure 2 ) showed that the treated material still retained abundant collagen and polysaccharide components. Scanning electron microscopy (SEM) observation results ( Figure 1 D) shows that the layered structure of collagen fibers inside the amniotic membrane remained stable before and after treatment and did not undergo significant changes.

[0079] High transparency is a key performance indicator of corneal repair materials. The human cornea exhibits a transmittance of more than 95% in the 400-800nm ​​spectral range due to its highly ordered arrangement of collagen fibers. The present invention evaluates the light transmittance of the material by UV-visible spectroscopy analysis ( Figure 1 E and Figure 3 ). The results showed that the transmittance of DAM and CAM increased after decellularization and cross-linking. However, the transmittance of DAM still does not meet the requirements of corneal application, which is only 82%. The transmittance of CAM after cross-linking increases with the increase of wavelength, and finally reaches 96%, which is comparable to the transparency of human cornea (higher than 95%). Material thickness measurement results ( Figure 4 ) showed that the thickness of FAM, DAM and CAM were 156±14.04μm, 131.33±14.57μm and 96±9.86μm, respectively. The decrease in DAM thickness was mainly attributed to the removal of the epithelial cell layer, while the decrease in CAM thickness was related to the enhanced aggregation of collagen molecules during the cross-linking process. The results of water content determination ( Figure 5 ) showed that the water content of CAM is comparable to that of FAM and DAM, and both can meet the 78% water content requirement of the natural cornea. This characteristic is beneficial to maintaining the water balance of the ocular surface and the exchange of nutrients. The mechanical properties test results show that ( Figure 1 F and Figure 6 ), CAM exhibits excellent mechanical properties. Stress-strain curve analysis shows that CAM can withstand greater loads than DAM and has a higher elastic modulus, and even some properties are better than FAM. Tensile strength test results ( Figure 1 G) It was further confirmed that after chemical cross-linking treatment, the tensile strength of CAM was significantly improved to a level comparable to that of FAM, which is beneficial for surgical operations. The above results show that cross-linking treatment effectively improves the problem of mechanical property degradation caused by decellularization while maintaining the original structure and properties of FAM.

[0080] Figure 1 The FTIR spectra shown in H reveal the presence of unique amide peaks (amide I, II, III, A, and B) in FAM, DAM, and CAM, which correspond to the triple helical structure of collagen. Specifically, all three materials have a peak at 1229 cm -1 The characteristic bands on the left and right correspond to NH deformation (amide III), 1542 cm -1 Corresponding to NH deformation (amide II), 1649 cm -1 Corresponds to C=O stretching (amide I). In addition, 3080 cm -1 and 3310cm -1The peaks at are attributed to amide B and amide A, respectively, mainly reflecting the stretching of NH and hydrogen-bonded OH groups. These findings indicate that decellularization and cross-linking treatments did not significantly change the conformation of collagen in the amniotic membrane. The peak intensity of DAM was lower than that of FAM, indicating that decellularization may damage the chemical bonds associated with the triple helix structure of collagen. In contrast, the increase in peak intensity observed in CAM indicates that the triple helix structure of collagen becomes more stable and ordered during the cross-linking process. The thermal denaturation temperature (Td) is a key parameter for corneal repair materials because these materials must remain stable when the ocular environment, such as humidity, fluctuates. Figure 1 As shown in I, neither decellularization nor cross-linking significantly altered the thermal stability of amniotic membrane in the DAM and CAM. Figure 1 J shows the degradation curve of the material in phosphate buffered saline (PBS) containing collagenase. The results showed that the degradation time of FAM was about 48 hours, while the degradation time of DAM was significantly shortened to 24 hours. However, after the cross-linking process, the degradation time of CAM was extended to about 120 hours. These findings indicate that chemical cross-linking improves the resistance of CAM to enzymatic degradation, thereby providing a more stable environment for corneal repair.

[0081] 2.2. Preparation and biomimetic enzyme activity of Mn-C dots and cross-linked decellularized bovine amniotic membrane composite (CDs@CAM)

[0082] Manganese carbon dots (Mn CDs) were successfully prepared by a solvothermal method, i.e., glutathione (GSH) and manganese chloride (MnCl 2 ) was dissolved in formamide and Mn CDs were synthesized by solvothermal reaction. Subsequently, cross-linked decellularized amniotic membrane (CAM) was immersed in MnCDs solution to prepare the composite material CDs@CAM ( Figure 7 A). Material characterization results show ( Figure 8 ), transmission electron microscopy (TEM) results and particle size distribution analysis results show that Mn CDs with an average diameter of 2.31±0.07nm were successfully synthesized. X-ray photoelectron spectroscopy (XPS) full spectrum analysis further verified that the chemical composition of Mn CDs mainly consists of C, N, O, and Mn elements. Scanning electron microscopy (SEM) element mapping image ( Figure 7 B) shows that Mn CDs are evenly distributed on the CAM surface. Fourier transform infrared spectroscopy (FTIR) analysis results ( Figure 7 C) showed that the introduction of Mn CDs did not change the collagen conformation of CAM, confirming the successful preparation of the composite material. In order to evaluate the effect of the introduction of Mn CDs on the performance of CAM, the transmittance, thermal stability and in vitro degradability of the composite material were systematically tested. The results showed that ( Figure 7D to 7F), the introduction of Mn CDs did not significantly change the transmittance, thermal stability and in vitro degradation properties of CAM, indicating that this modification method successfully achieved functional modification while maintaining the excellent optical properties of CAM.

[0083] In order to explore the multi-enzyme-like activity introduced by Mn CDs in CDs@CAM composites, we aimed to effectively remove overexpressed reactive oxygen species (ROS) in the microenvironment during infection and inflammation. 2 ·-), hydroxyl radicals (·OH) and hydrogen peroxide (H 2 O 2 ) are three typical ROS. Figure 7 As shown in G, the multi-enzyme activity of CDs@CAM may be catalyzed by O 2 -Converted to H 2 O 2 and O 2 , and H 2 O 2 Decompose into O 2 and H 2 O, and can also effectively remove ·OH. The hydroxyl radical scavenging activity (HRSA), superoxide dismutase (SOD) activity and catalase (CAT) activity of CDs@CAM were measured respectively. Figure 7 As shown in Figure 1, the HRSA results measured by UV-Vis spectrophotometry using 5,5'-tetramethylbenzidine (TMB) as a probe showed that the scavenging rates of ·OH in CDs@CAM groups with different Mn CDs contents (42.39±4.49%, 69.12±5.80%, and 84.90±5.31%, respectively) were significantly higher than those in the pure CAM group (4.13±1.88%), and the scavenging effect increased with the increase in the Mn CDs loading. In addition, the presence of hydroxyl radical scavenging activity was further confirmed by electron spin resonance (ESR) analysis using 5,5-dimethyl-1-pyrrolidine n-oxide (DMPO) as a capture agent ( Figure 7 I). The SOD enzyme activity of CDs@CAM was evaluated using a SOD enzyme activity detection kit and ESR technology ( Figure 7 J to 7K), the results showed that compared with CAM, CDs@CAM exhibited significant SOD-like enzyme activity and could effectively remove O 2 ·-. The remaining H 2 O 2 The results showed that CDs@CAM has high efficiency in removing H 2 O 2 The clearance effect was concentration-dependent ( Figure 7In addition to the above enzyme activities, the scavenging activity of CDs@CAM against representative reactive nitrogen species (RNS) was also evaluated. The scavenging rate of CDs@CAM (96 μg / ml) was 76.36 ± 2.24 ( Figure 7 M). Considering the high biomimetic enzyme activity of CDs@CAM and minimizing the impact on its transparency, the subsequent experiments of the present invention used a concentration of 60 μg / ml CDs@CAM to carry out cell and animal experiments to explore its antioxidant and anti-inflammatory effects in vitro and in vivo.

[0084] 2.3 Cytocompatibility, antioxidant and anti-inflammatory properties of Mn-C dots and cross-linked decellularized bovine amniotic membrane composites (CDs@CAM)

[0085] Live / dead staining and CCK-8 method ( Fig. 9 ) evaluated the biocompatibility of CDs@CAM with human corneal epithelial cells (HCECs). After 1, 3, and 5 days of co-culture, live / dead staining images showed that HCECs continued to proliferate in all experimental groups. Most cells showed green fluorescence, indicating living cells, while the number of dead cells showing red fluorescence was very small. CCK-8 assay further showed that CDs@CAM supported cell proliferation, and cell viability remained high after 5 days of culture. These findings provide evidence for the good biocompatibility of CDs@CAM.

[0086] Based on the pathological characteristics of alkali-burned cornea, HCECs were used to establish H 2 O 2 The induced oxidative stress model was used to study the antioxidant effect of CDs@CAM. 2',7'-dichlorofluorescein diacetate (DCFH-DA) was used as a fluorescent probe to detect the ROS scavenging effect of each group of materials on HCECs cells. Fig.10 A and Fig.10 As shown in B, H 2 O 2 Clear green fluorescence appeared in both the CDs@CAM group and the CDs@CAM group. However, there was almost no green fluorescence in the CDs@CAM group, indicating that intracellular ROS were cleared by multiple enzyme activities.

[0087] Studies have shown that oxidative stress is a direct result of alkali burns, and immediate removal of ROS can inhibit pathological corneal neovascularization (CNV). Excessive production of ROS leads to the release of calcium ions, which in turn promotes further production of ROS and exacerbates inflammation. Given that the antioxidant properties of CDs@CAM have been confirmed by its effective removal of ROS, we next conducted a series of anti-inflammatory experiments to evaluate the anti-inflammatory efficacy of CDs@CAM. It is known that activated macrophages (RAW 264.7) exhibit heterogeneous cell populations (M1, M2, etc.), polarization toward M1 has a stronger pro-inflammatory tendency, while polarization toward M2 has the property of reducing inflammation. Therefore, RAW 264.7 cells were stimulated with lipopolysaccharide (LPS) and co-cultured with each group of materials, and then labeled with antibodies CD86 (a marker of M1 macrophages) and CD206 (a marker of M2 macrophages), and the phenotype of RAW 264.7 cells was measured by flow cytometry. From Fig.10 As can be seen in C, the M1 type increased significantly after LPS stimulation, indicating the worsening of the inflammatory response. After adding material treatment, compared with the CAM group, the M1 type of CDs@CAM decreased and the M2 type increased, proving that CDs@CAM has a high anti-inflammatory effect. Immunofluorescence experiments of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were performed to qualitatively analyze the inflammatory response that occurred. Fig.10 The immunofluorescence staining results in D showed the expression of multiple inflammatory factors in the cells. The LPS group and the CAM group showed obvious green fluorescence, while the fluorescence intensity of the CDs@CAM group was relatively weak. To further verify the anti-inflammatory properties of CDs@CAM, the culture medium of RAW 264.7 cells after treatment was taken and the levels of inflammatory factors were quantitatively detected using an enzyme-linked immunosorbent assay (ELISA) kit ( Fig.10 E to 10G). The overall trend of the results was consistent with the above immunofluorescence detection results.

[0088] In summary, CDs@CAM can effectively scavenge ROS and reverse the pro-inflammatory phenotype of macrophages, all of which are related to the pathological mechanism of alkali-burned cornea. This result encouraged us to further study its therapeutic effect in vivo.

[0089] 2.4 Manganese carbon dots and cross-linked acellular bovine amniotic membrane composite (CDs@CAM) for epithelialization and corneal reconstruction of alkali-burned cornea

[0090] In order to detect the antioxidant, anti-inflammatory and therapeutic effects of CDs@CAM in corneal repair, a corneal alkali burn model was constructed in SD rats and the rats were randomly divided into four groups: a normal group without any injury, a control group without any treatment, and a CAM or CDs@CAM treatment group. Fig.11A shows the timeline of animal experiments, including the time points of each experimental operation and the specific methods of postoperative examination. After the corneal alkali burn model of SD rats was successfully established, CAM and CDs@CAM were used for treatment for 28 days, respectively, and the treatment process was tracked by slit lamp and continuous anterior segment optical coherence tomography (AS-OCT). Seven days after surgery, rats from the four groups were randomly selected for neck resection and enucleation, and H&E staining and immunofluorescence staining of inflammatory cytokines were performed. After 28 days of treatment, all remaining SD rats underwent neck resection and enucleation, fixed with formalin, and subjected to H&E staining and immunofluorescence staining of corneal function-related indicators. Corneal images taken by slit lamp microscope ( Fig.11 B) showed that alkali burns occurred in all groups. After the material intervention, the corneal edema in the CDs@CAM group was significantly reduced, and the transparency of the cornea also recovered to normal levels at a faster rate. The slit lamp images stained with sodium fluorescein showed that both CAM and CDs@CAM could effectively accelerate wound healing, but the healing speed of corneal epithelial defects in the CDs@CAM group was faster than that in the CAM group. Statistical analysis results of corneal epithelial healing rate ( Fig.11 D) further confirmed the above conclusion. In addition, corneal thickness is an important parameter for evaluating the degree of corneal damage and subsequent repair. Fig.11 C) showed that both CAM and CDs@CAM significantly promoted corneal tissue repair, but CDs@CAM showed better effects in reducing corneal edema and restoring corneal thickness. Fig.11 As shown in E, the corneal thickness of the CDs@CAM treatment group decreased rapidly and returned to normal levels (below 200 μm) on day 7. Importantly, the results showed that although the CAM treatment group promoted corneal recovery after acute alkali burn, its effectiveness was limited compared with the CDs@CAM group, which combined the dual therapeutic advantages of CAM and Mn CD. Therefore, CDs@CAM is a promising therapeutic agent that can accelerate the complete recovery of corneal transparency, thickness, and epithelial integrity after alkali burns.

[0091] 2.5. Histological evaluation of the anti-inflammatory effect of manganese carbon dots and cross-linked acellular bovine amniotic membrane composite (CDs@CAM) on alkali-burned cornea in vivo

[0092] After alkali burn, oxidative stress in the cornea increased significantly, leading to a significant inflammatory response. The multiple enzyme activities of CDs@CAM can clear excess ROS in the alkali burn site, thereby restoring the balance of antioxidant and pro-oxidative mechanisms in the cornea and reducing the occurrence of inflammation. Therefore, we studied the expression of relevant classical inflammatory cytokines (TNF-α, IL-1β and IL-6) in the cornea in the early stage after intervention with different treatments. H&E staining and immunofluorescence staining were performed on the cornea of ​​SD rats on the 7th day. Fig.12As shown, H&E staining showed that the corneal thickness of the control group was uneven, inflammatory cell infiltration was obvious (black arrow), and there were defects in both the corneal epithelium and endothelium (red arrow). Compared with the control group, CAM treatment significantly reduced the infiltration of inflammatory cells, but some defects in the corneal epithelium still existed (red arrow). In contrast, the corneas in the CDs@CAM treatment group showed complete corneal structure and initial regeneration of a single layer of complete epithelial cell layer, with almost no infiltration of inflammatory cells. In addition, immunofluorescence staining of inflammatory cytokines (including TNF-α, IL-6, and IL-1β) was performed to evaluate the level of inflammation in vivo after treatment with each group of materials ( Fig.13 A). In the control group, the cornea was still in a state of strong inflammatory response, characterized by the presence of a large number of proinflammatory cytokines, which almost covered the entire cornea. This situation may cause corneal edema, further infiltration of inflammatory cells, prolonged healing time, and damage to corneal tissue structure. In the CAM treatment group, the expression of these inflammatory cytokines was relatively reduced. However, there was almost no red immunofluorescence in the CDs@CAM group, indicating that the corneal inflammatory response had subsided. These inflammatory cytokines mainly promote the aggregation and activation of inflammatory cells and enhance the release of inflammatory mediators, thereby disrupting the corneal healing process. In the corneal inflammatory response, TNF-α stimulates fibroblast proliferation, promotes scar tissue formation, and hinders visual recovery. IL-1β can induce corneal endothelial cell apoptosis, activate corneal fibroblasts and IL-6, thereby amplifying inflammatory signals at the molecular level and promoting the infiltration of inflammatory cells into the corneal stroma. Quantitative fluorescence intensity analysis results ( Fig.13 B to 13D) further demonstrated that CDs@CAM exhibited the most significant anti-inflammatory effect. Because the decreased expression levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) were associated with decreased ROS levels, the reduction in inflammatory cytokines observed in the CDs@CAM group may be attributed to the multi-enzyme activity of CDs@CAM in eliminating ROS.

[0093] 2.6. Histological evaluation of the repair effect of manganese carbon dots and cross-linked acellular bovine amniotic membrane composite (CDs@CAM) on alkali-burned cornea

[0094] After 28 days of treatment, corneal tissue sections of Sprague-Dawley (SD) rats were subjected to morphological analysis and immunofluorescence staining of corneal function indicators. Fig.14H&E staining in A shows that the cornea of ​​the control group was thicker than that of the CAM and CDs@CAM treated groups. In addition, there was a significant loss of corneal endothelial cells (red arrows). Severe inflammation within the cornea can lead to the loss of corneal endothelial cells, which are non-regenerative. This defect indicates that the Control group experienced a severe inflammatory response. In contrast, the corneal morphology of the CAM group was almost restored, but the arrangement of its matrix collagen fibers appeared relatively chaotic. The corneal structure and thickness of the CDs@CAM group were restored to a healthy state, showing a clear hierarchical organization. Further quantitative analysis of corneal epithelial thickness was performed ( Fig.14 B), the results showed that only the corneal epithelial thickness of the CDs@CAM group returned to normal levels. Immunofluorescence staining results of the tight junction protein zonula occludens-1 (ZO-1) in the corneal epithelium ( Fig.14 C) and corresponding quantitative data analysis ( Fig.14 D) The results showed that the expression of ZO-1 protein in the CDs@CAM group was comparable to that in the normal group and significantly higher than that in the Control group and the CAM group. ZO-1 protein is essential for maintaining the structural integrity and barrier function of the corneal epithelium. Therefore, the restoration of ZO-1 protein in the corneal epithelium of the CDs@CAM group further proves that CDs@CAM is beneficial to the restoration of corneal structural integrity and barrier function. Immunofluorescence image ( Fig.14 C) showed that both the CAM group and the Control group had a certain level of CD31 expression, while no expression was detected in the CDs@CAM group and the normal group. CD31 is a platelet endothelial cell adhesion molecule that is often associated with corneal neovascularization (CNV). Therefore, the reduced expression of CD31 suggests that CDs@CAM may inhibit the generation of corneal neovascularization (CNV) to a certain extent. Quantitative results analysis of histological evaluation ( Fig.14 Furthermore, given that sustained fibroblast activation could lead to corneal scarring, immunofluorescence staining of α-smooth muscle actin (α-SMA) in the stroma was performed to assess fibroblast formation and corneal scarring. Fig.14 C and Fig.14 The immunofluorescence images and quantitative data shown in F confirmed the expression of α-SMA in both the CAM group and the Control group, which may be due to the enhanced inflammatory response in these groups. However, no obvious α-SMA expression was observed in the CDs@CAM group, indicating that fibroblast activation was not induced. Therefore, CDs@CAM supports the formation of tight junctions in the regenerated epithelial cell layer and can inhibit corneal neovascularization (CNV) and scar formation.

[0095] In summary, the results of the study on corneal repair after alkali burns showed that CAM had limited effect in treating alkali burn injuries. Without loading of Mn CDs, CAM had a weak ability to scavenge ROS and exert anti-inflammatory effects at the alkali burn site, resulting in a significant corneal inflammatory reaction on the 7th day, and the anti-scarring and anti-neovascular effects of the CAM group were significantly lower than those of the CDs@CAM group on the 28th day. The addition of Mn CDs significantly enhanced the antioxidant and anti-inflammatory effects of CAM. When applied to the alkali burn site, CDs@CAM could effectively exert its multi-enzyme activity to scavenge excess ROS to maintain intraocular oxidative stress homeostasis. At the same time, it also inhibited the activation of NF-κB by excessive ROS, thereby reducing the release of inflammatory cytokines. Therefore, CDs@CAM could significantly reduce corneal inflammation and edema and promote rapid repair of the corneal epithelium. These findings emphasize that CDs@CAM has corneal repair ability as well as antioxidant and anti-inflammatory properties, which are attributed to the presence of bioactive substances and multi-enzyme activities in the material. This suggests that CDs@CAM may be a promising candidate material for the treatment of alkali-burned cornea.

[0096] 3. Conclusion

[0097] The present invention successfully prepared CAM with biocompatibility and excellent physical properties (high transmittance, high thermal stability, excellent mechanical properties and resistance to enzymatic degradation) through decellularization and cross-linking treatment. In addition, we innovatively developed Mn CDs with multi-enzyme activity and composited it with CAM (obtaining CDs@CAM). The resulting composite material CDs@CAM not only has the multi-enzyme activity unique to Mn CDs, but also retains the excellent physical properties and biocompatibility of CAM. In vitro enzyme activity assays showed that the CDs@CAM composite material has significant CAT-like enzyme activity, SOD-like enzyme activity, and ·OH and RNS scavenging capabilities. Cell experiments showed that CDs@CAM has good cell compatibility. In H 2 O 2 In experiments with stimulated HCECs and LPS-stimulated RAW 264.7 macrophages, CDs@CAM significantly reduced intracellular ROS levels and alleviated inflammatory responses, showing significant antioxidant and anti-inflammatory properties. CDs@CAM was used to repair alkali-burned corneas, and the results showed that it was very effective in promoting corneal epithelial repair and exhibited good anti-inflammatory effects in vivo. After treatment, corneal thickness and clarity returned to normal levels. These findings suggest that CDs@CAM is expected to become a promising candidate material for the treatment of alkali-burned corneas and corneal epithelial injuries and diseases. This simple and effective material surface modification method also has the potential to be widely used in optical and implant materials to endow them with various biological functions.

Claims

1. The use of cross-linked decellularized amniotic membrane (CAM) and manganese carbon dots (Mn CDs) in the preparation of ophthalmic medical materials for repairing alkali-burned cornea.

2. The use according to claim 1, characterized in that: The mass ratio of cross-linked decellularized bovine amniotic membrane (CAM) to manganese carbon dots (MnCDs) is: Cross-linked decellularized bovine amniotic membrane (CAM): manganese carbon dots (Mn CDs) = 374-1499:

1.

3. The use according to claim 2, characterized in that: The mass ratio of cross-linked decellularized bovine amniotic membrane (CAM) to manganese carbon dots (MnCDs) is: Cross-linked decellularized bovine amniotic membrane (CAM): manganese carbon dots (Mn CDs) = 599:

1.

4. The use according to any one of claims 1 to 3, characterized in that: The ophthalmic medical material promotes corneal epithelial regeneration and inhibits the formation of new blood vessels and scars.

5. A composite for repairing alkali-burned cornea, characterized in that: It is compounded from the following raw materials in the following weight ratio: Cross-linked decellularized bovine amniotic membrane (CAM) 375-1500 parts, manganese carbon dots (Mn CDs) 1 part.

6. The composite for repairing alkali-burned cornea according to claim 5, characterized in that: It is compounded from the following raw materials in the following weight ratio: 599 parts of cross-linked decellularized bovine amniotic membrane (CAM) and 1 part of manganese carbon dots (Mn CDs).

7. The composite for repairing alkali-burned cornea according to claim 5 or 6, characterized in that: The manganese carbon dots (Mn CDs) are prepared by solvent thermal synthesis, and the specific preparation method is as follows: Manganese dichloride (MnCl2) and glutathione (GSH) are completely dissolved in formamide; then the mixture is placed in a polytetrafluoroethylene-lined hot liquid autoclave to react at high temperature and high pressure to generate manganese carbon dots (Mn CDs); the mass ratio of manganese dichloride (MnCl2) to glutathione (GSH) is 1:6.35; the reaction temperature is 180°C and the reaction time is 4 hours; After the reaction, the solution was cooled to room temperature for filtration and dialyzation, and then concentrated using a rotary evaporator to prepare Mn CDs.

8. The method for preparing the composite for repairing alkali-burned cornea according to any one of claims 5 to 7, characterized in that: It includes the following steps: a. Weigh the raw materials of each weight ratio; b. The cross-linked decellularized amniotic membrane (CAM) was immersed in a manganese carbon dots (Mn CDs) solution and placed on an oscillator running at 100 rpm for 48 hours.

9. Use of the complex according to any one of claims 5 to 7 in the preparation of an ophthalmic medical material for repairing alkali-burned cornea.

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