In-situ oxygen-releasing collagen-based hydrogel eye drop with cascade enzyme activity and preparation method and use thereof

Abstract

This invention provides an in-situ oxygen-releasing collagen-based hydrogel eye drop with cascade enzyme activity, its preparation method, and its uses. The in-situ oxygen-releasing collagen-based hydrogel eye drop provided by this invention contains manganese-curcumin carbon dots (MC-CDs), which are prepared using curcumin as the carbon source and manganese ions as the metal dopant raw material via an alkali-assisted hydrothermal carbonization method. The mass ratio of manganese ions to curcumin is 8-9:100. This invention also provides an ophthalmic pharmaceutical composition for treating corneal alkali burns, which is an ophthalmic preparation made from the aforementioned manganese-curcumin carbon dots (MC-CDs) and collagen. This invention constructs a collagen-based nanoenzyme composite hydrogel eye drop (CG@MC) integrating high light transmittance, long-lasting ocular surface retention, dynamic immune regulation, and in-situ oxygen release function. This system can effectively block the vicious cycle of oxidative stress, persistent inflammation, and tissue hypoxia microenvironment after corneal alkali burns. As a non-invasive drug delivery treatment, this eye drop provides a practical solution for the emergency treatment and long-term postoperative management of corneal chemical injuries, and is of great significance for the clinical translation of corneal regenerative medicine.

Description

Technical Field

[0001] This invention relates to an in-situ oxygen-releasing collagen-based hydrogel eye drop with cascade enzyme activity, an ophthalmic pharmaceutical composition for treating corneal alkali burns, and its preparation method and uses. Background Technology

[0002] Alkali burns of the cornea are a significant cause of corneal blindness in clinical practice. Following an alkali burn, the corneal tissue often enters a state of persistent inflammation and hypoxia, which activates hypoxia-inducible factor (HIF-1α) and its downstream vascular endothelial growth factor (VEGF) pathway, inducing corneal neovascularization (CNV). Simultaneously, excessive accumulation of reactive oxygen species (ROS) in the inflammatory microenvironment promotes the transformation of corneal fibroblasts into myofibroblasts, leading to abnormal collagen fiber deposition and scar tissue formation, ultimately resulting in loss of corneal transparency. The physiological barrier characteristics of the cornea, such as tear flushing, blink reflex, and the tight junctions of the corneal epithelium, result in the short retention time and low bioavailability of traditional eye drops on the ocular surface, making it difficult to achieve consistently effective therapeutic concentrations.

[0003] There are currently some literature reports on the treatment of corneal alkali burns with curcumin, such as Li Yan, Qin Li, Li Pinming. Experimental study on the treatment of corneal alkali burns in rats with curcumin [J]. Journal of Hunan University of Traditional Chinese Medicine, 2015, 35(8): 20-24. This literature only uses curcumin solution as a single raw material to exert limited anti-inflammatory and anti-apoptotic effects through invasive injection. Zhang Weili, Wu Zhihong, Du Lina, et al. Treatment of rabbit corneal alkali burns with curcumin-chitosan liposomes [J]. International Journal of Pharmaceutical Research, 2016, 43(4): 705-710. This literature uses liposome technology to encapsulate curcumin and then uses chitosan coating with positive charge to enhance corneal retention. It utilizes curcumin itself to inhibit VEGF to reduce corneal neovascularization and has anti-inflammatory and antioxidant effects. Cui Yuexin. Experimental study on the effect of iron-curcumin metal nanocomplex on corneal neovascularization after alkali burn in rats [D]. Jinzhou: Jinzhou Medical University, 2022. This literature reports the formation of metal complexes (Fe-Cur CPNs) by iron ions and curcumin, which are mainly solubilized by PVP. Fe-Cur CPNs can be administered by subconjunctival injection and can have anti-inflammatory and anti-angiogenic effects, mainly relying on simple antioxidant and inhibition of the NF-κB pathway.

[0004] In ophthalmic clinical treatment, subconjunctival injection is a minimally invasive periocular drug delivery method, but it carries risks such as local tissue damage, uncontrollable drug distribution, systemic absorption leading to systemic toxicity, and the need for frequent repeat injections. Topical eye drops remain the most widely used method. Traditional eye drops typically dissolve or disperse the active pharmaceutical ingredient in an aqueous medium and are applied to the ocular surface via instillation, spreading to all layers of the cornea with the help of tears to achieve therapeutic effects. Typical examples of this type of preparation include antibiotic eye drops, anti-inflammatory eye drops, and artificial tears. The administration process is simple, can be performed by the patient themselves, and requires no special equipment.

[0005] Hydrogels, as materials with excellent biocompatibility, have shown great application potential in the field of corneal repair by enhancing drug retention properties on the corneal epithelium and reducing toxic side effects. In recent years, thermosensitive in-situ gel drug delivery systems, represented by poloxamer F127, have been extensively studied for ocular drug delivery. These formulations are in a free-flowing liquid state at room temperature, facilitating ocular instillation; upon contact with the ocular surface (approximately 35°C), they rapidly undergo a sol-gel phase transition, forming a semi-solid gel, thereby prolonging the drug's retention time on the corneal surface.

[0006] Traditional eye drops have an extremely short retention time on the ocular surface (usually <5 minutes), and their bioavailability is generally below 5%. Patients need to administer the drops frequently to maintain an effective drug concentration, which not only increases the risk of ocular irritation but also leads to decreased patient compliance. Furthermore, traditional eye drops lack the ability to actively regulate complex pathological microenvironments (such as hypoxia and oxidative stress), making it difficult to effectively break the vicious cycle of "hypoxia-inflammation-angiogenesis" after alkali burns. While thermosensitive hydrogels prolong the ocular surface retention time, they face the challenge of poor biomechanical compatibility in practical applications. Thermosensitive polymers, such as poloxamer, often exhibit high mechanical rigidity after phase transitions, making it difficult to perfectly match the complex dynamic curvature of the ocular surface and the shear stress generated by frequent blinking. This mechanical mismatch may lead to increased foreign body sensation, ocular irritation, and even damage to newly formed epithelium. In addition, these materials typically lack bioactive functions, acting only as passive delivery carriers. They cannot actively regulate the redox homeostasis of the wound or improve the hypoxic microenvironment, lack in-situ oxygen release capabilities, and cannot fundamentally improve the hypoxic microenvironment or inhibit angiogenesis. Furthermore, most materials lack sufficient optical transparency (such as traditional collagen hydrogels with a light transmittance of only about 70%), which may interfere with the cornea's "optical window" function. Summary of the Invention

[0007] This invention provides manganese-curcumin carbon dots (MC-CDs), an ophthalmic pharmaceutical composition for treating corneal alkali burns, and its preparation method and uses.

[0008] This invention provides a manganese-curcumin carbon dot MC-CDs, which are prepared by using curcumin as a carbon source and manganese ions as a metal dopant raw material, and by an alkaline-assisted hydrothermal carbonization method. The mass ratio of manganese ions to curcumin is 8-9:100.

[0009] The mass ratio of manganese ions to curcumin is 8.73:100.

[0010] The manganese ions are divalent or trivalent manganese ions, including manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; the particle size range of the manganese-curcumin carbon dots MC-CDs is 2.6-5.9 nm.

[0011] The raw materials also include one of formamide, methanol, acetamide, and ethylene glycol, with a mass-to-volume ratio of 1:2 to curcumin.

[0012] This invention also provides a method for preparing the aforementioned manganese-curcumin carbon dots MC-CDs, which includes the following steps:

[0013] a. Add curcumin to formamide and add NaOH as a solubilizer to prepare a precursor solution;

[0014] b. Pretreatment: Manganese chloride was slowly introduced into the precursor solution under constant speed magnetic stirring and homogenized stirring was maintained for 30 min.

[0015] c. Hydrothermal carbonization: The mixture is transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 4 hours.

[0016] d. Purification: After the reaction system cooled naturally to room temperature, the product was filtered through a 0.22 μm microporous membrane and then dialyzed with a molecular weight cutoff of 1000 Da. The dialysis process was carried out in ultrapure water for 72 h, with water changed every 6 h to ensure that impurities were completely removed.

[0017] e. Product collection: The dark green solid powder is collected by vacuum freeze-drying technology and sealed and stored in a desiccator.

[0018] The present invention also provides the use of the aforementioned manganese-curcumin carbon dots MC-CDs in the preparation of ophthalmic drugs for treating corneal alkali burns. Alternatively, the use of the aforementioned manganese-curcumin carbon dots MC-CDs in the preparation of topical drugs with antioxidant and anti-inflammatory effects.

[0019] This invention provides an ophthalmic pharmaceutical composition for treating corneal alkali burns. It is an ophthalmic preparation made from manganese-curcumin carbon dots (MC-CDs) as described in any one of claims 1-4 and collagen as raw materials. The mass ratio of manganese-curcumin carbon dots (MC-CDs) to collagen is 1:2 to 1:5, and the optimal mass ratio is 1:4.

[0020] The ophthalmic preparations mentioned above are eye drops, eye ointments, and ophthalmic gels.

[0021] The present invention also provides a method for preparing the ophthalmic pharmaceutical composition for treating corneal alkali burns, wherein the method for preparing the eye drops includes the following steps: a. Dissolve collagen in glacial acetic acid solution to prepare a collagen solution, and add an equal volume of PBS solution;

[0022] b. Dissolve manganese-curcumin carbon dots (MC-CDs) in 1% glycerol;

[0023] c. Add the MC-CDs / glycerol solution prepared in step a to the collagen / acetic acid solution prepared in step a, adjust the pH to 7.4 with sodium hydroxide, and place at 37°C to obtain the eye drops of the present invention.

[0024] The present invention also provides the use of the ophthalmic pharmaceutical composition in the preparation of a medicament for treating corneal alkali burns.

[0025] This invention provides an in-situ oxygen-releasing collagen-based hydrogel eye drop system with cascade enzyme activity, which is an intelligent ocular drug delivery system. By combining a cascade enzyme catalytic system with a collagen-based hydrogel carrier, it achieves the dual functions of ocular oxygen delivery and drug delivery. During use, the in-situ oxygen-releasing collagen-based hydrogel eye drop system gels upon instillation, continuously releasing oxygen in situ on the ocular surface through an enzyme-catalyzed cascade reaction. Simultaneously, the collagen hydrogel carrier enables sustained drug release and tissue repair, making it particularly suitable for treating corneal alkali burns.

[0026] This invention transforms curcumin into manganese-doped carbon dots (MC-CDs) with SOD / CAT-like multi-enzyme activity, and constructs a hydrogel delivery system (CG@MC) with high transparency, shear-thinning properties, and long-lasting retention on the ocular surface by regulating collagen self-assembly with glycerol. Mechanistically, this invention expands from simple anti-inflammatory and anti-apoptotic effects to a multi-synergistic repair mechanism involving "antioxidation, in-situ oxygen release, immunomodulation, and inhibition of scars / vascularity". In clinical translation, this invention innovates invasive injection into non-invasive ophthalmic drop administration, improving patient compliance and clinical feasibility.

[0027] The manganese-doped curcumin carbon dot composite collagen hydrogel eye drops (CG@MC) constructed in this invention utilizes glycerol to regulate collagen self-assembly, endowing the hydrogel with high transparency and shear-thinning properties, significantly prolonging the ocular surface retention time to over 40 minutes. Furthermore, the synthesized MC-CDs not only retain the surface groups of curcumin, enabling it to exert its antioxidant properties, but also utilize Mn... 2+ / Mn 3+ Doping endows it with SOD / CAT-like multiple enzyme activity, which can efficiently remove ROS and produce oxygen in situ, improving the hypoxic microenvironment after alkali burns from the source. The system can also regulate the polarization of macrophages from M1 to M2, realize the remodeling of the immune microenvironment, thereby synergistically inhibiting angiogenesis, reducing fibrosis and promoting the restoration of corneal transparency. It shows better comprehensive efficacy than traditional liposomes in terms of promoting healing, anti-inflammation and anti-scarring.

[0028] This invention, CG@MC, is administered via non-invasive eye drops. It achieves high light transmittance and over 40 minutes of ocular surface retention through glycerol-regulated collagen hydrogel, overcoming the drawbacks of invasive injection. Functionally, CG@MC not only possesses broad-spectrum antioxidant capabilities but also releases oxygen in situ through the multi-enzyme cascade catalytic activity of manganese, alleviating the hypoxic microenvironment at its source. Simultaneously, it exerts multiple synergistic effects of anti-inflammation, anti-scarring, and immune remodeling, resolving the vicious cycle of "hypoxia-inflammation-scarring-neovascularization" after alkali burns. In clinical translation, it offers superior safety and convenience.

[0029] The beneficial effects of this invention are:

[0030] This invention presents a collagen-based nanoenzyme composite hydrogel eye drop (CG@MC) that integrates high light transmittance, long-lasting ocular surface retention, dynamic immune regulation, and in-situ oxygen release. This system effectively blocks the vicious cycle of oxidative stress, persistent inflammation, and tissue hypoxia following corneal alkali burns. As a non-invasive treatment, this eye drop provides a practical solution for the emergency treatment and long-term postoperative management of corneal chemical burns, and is of great significance for the clinical translation of corneal regenerative medicine. Attached Figure Description

[0031] Figure 1 A schematic diagram of the synthesis and post-processing of MC-CDs;

[0032] Figure 2 Characterization of MC-CDs: (a) TEM image; (b) particle size distribution histogram;

[0033] Figure 3 Structural characterization of MC-CDs: (a) Raman spectrum; (b) XRD spectrum; (c) FTIR spectrum;

[0034] Figure 4XPS characterization of MC-CDs: (a) full spectrum; (b) high-resolution Mn spectrum; (c) high-resolution O spectrum; (d) high-resolution C spectrum;

[0035] Figure 5 Characterization of the antioxidant properties of MC-CDs: (a) scavenging ability of ABTS; (b) scavenging ability of DPPH; (c) TMB method for detecting the scavenging ability of MC-CDs for ·OH; (d) evaluation of the scavenging ability of ·OH using electron spin resonance (ESR).

[0036] Figure 6 Enzyme-like activity characterization of MC-CDs: (a) SOD-like enzyme activity; (b) O2•- scavenging capacity assessed using electron spin resonance (ESR); (c) CAT-like enzyme activity; (d) oxygen release capacity assay.

[0037] Figure 7 The image shows the process of CG hydrogel being dripped from the dropper bottle, and a top view showing the light transmittance;

[0038] Figure 8 The rheological properties of the CG hydrogel are shown in the following figures: (a) viscosity curve; (b) frequency scan; and (c) shear-creep recovery test, simulating human blinking. The shear stress during the blinking phase was set to 17.5 Pa and lasted for 0.3 s, while the shear stress during the recovery phase was set to 0 Pa and lasted for 4 s, simulating the subsequent eye-opening recovery time.

[0039] Figure 9 For the performance characterization of CG hydrogel: (a) transmittance test; (b) molecular simulation analysis of conformational changes in the interaction between glycerol and collagen molecules; (c) changes in the number of hydrogen bonds between collagen molecules; (d) changes in the number of hydrogen bonds between collagen and glycerol.

[0040] Figure 10 Evaluation of the retention performance of MC-CDs and CG@MC in the eye: (a) Optical coherence tomography (OCT) monitoring images; (b) Evaluation of ocular surface retention behavior based on sodium fluorescein tracer;

[0041] Figure 11 FDA / PI stained images of materials and HCECs after 3 days of co-incubation;

[0042] Figure 12 Quantitative analysis of cell viability after co-incubation of materials and HCECs for 3 days;

[0043] Figure 13(a) Schematic diagram of the antioxidant experiment; (b) CLSM imaging of HCECs after DCFH-DA / Hoechst fluorescence staining; (c) Quantitative data of ROS scavenging rate; (d) CLSM imaging of intracellular oxygen detected by oxygen indicator [Ru(dpp)3]Cl2; (e) Statistical analysis of fluorescence intensity representing O2.

[0044] Figure 14 (a) Schematic diagram of exploring the ability of macrophage polarization to regulate inflammation; (b) CLSM imaging of RAW 264.7 after IL-6 / Hoechst fluorescence staining; (c) Quantitative data of IL-6 positive expression rate; (d) CLSM imaging of RAW 264.7 after IL-10 / Hoechst fluorescence staining; (e) Quantitative data of IL-10 positive expression rate;

[0045] Figure 15 (a) Images of corneal observation under slit lamp bright field; (b) Images of corneal epithelialization observed under cobalt blue light by fluorescein staining; (c) Images of corneal neovascularization 21 days postoperatively; (d) Statistical data on corneal epithelialization rate.

[0046] Figure 16 (a) OCT images of the corneal cross-section after alkali burn; (b) Statistical data on corneal thickness. Detailed Implementation

[0047] Example 1: Preparation of the eye drops of the present invention

[0048] a. Preparation of manganese-curcumin carbon dots (MC-CDs):

[0049] First, 100 mg of curcumin was added to 50 mL of formamide, and 50 μL of NaOH (5M) was added dropwise as a solubilizer. Under constant-speed magnetic stirring, 20 mg of manganese chloride (with a manganese ion to curcumin mass ratio of 8.73:100) was slowly introduced into the precursor solution, and homogeneous stirring was maintained for 30 min to establish a stable reaction system. Subsequently, the mixture was transferred to a polytetrafluoroethylene-lined high-pressure reactor and subjected to hydrothermal carbonization at 180 °C for 4 h. After the reaction system cooled naturally to room temperature, the product was sequentially filtered through a 0.22 μm microporous membrane and dialyzed with a molecular weight cutoff of 1000 Da. The dialysis process was carried out in ultrapure water for 72 h, with water changes every 6 h to ensure complete removal of impurities. Finally, the dark green solid powder was collected by vacuum freeze-drying and stored in a desiccated bottle.

[0050] b. Preparation of collagen-based hydrogel eye drops (CG@MC):

[0051] First, collagen was dissolved in 0.5 M glacial acetic acid solution to prepare a 4 mg / ml collagen solution, and then an equal volume of PBS solution was added. MC-CDs were pre-dissolved in 1% glycerol to a concentration of 1 mg / ml. An equal volume of the 1% glycerol solution containing MC-CDs was added to the 4 mg / ml collagen solution, and the pH was adjusted to 7.4 using 5 M sodium hydroxide. The solution was then incubated at 37°C for 30 min. The resulting composite hydrogel eye drops were named CG@MC.

[0052] Example 2 Characterization of MC-CDs of the present invention

[0053] MC-CDs are produced by solvothermal reaction of Mn in formamide. 2+ Synthesized by reacting with Cur ( Figure 1 Because Curl is rich in carbon and oxygen and has abundant functional groups, it not only acts as a carbon source for carbon dots in this preparation process, but also as a stabilizer and a reactant with Mn. 2+ Combining and promoting Mn 2+ Entering the carbon core of the carbon dot facilitates the formation of doped carbon dots. The nitrogen element contained in the solvent formamide can act as a nitrogen source, achieving a certain nitrogen doping effect.

[0054] The microstructure and particle size distribution of MC-CDs are as follows: Figure 2 As shown in the figure. TEM results indicate that the prepared carbon dots are uniformly dispersed, exhibiting an approximately spherical morphology, and their size distribution is relatively narrow. Quantitative statistical analysis of the spherical particles in the images using ImageJ software revealed that MC-CDs exhibit a narrow particle size distribution, mainly concentrated in the range of 2.6-5.9 nm. Further data fitting results indicate that the average particle size of the sample is 4.09 nm.

[0055] Raman spectroscopy ( Figure 3 a) Structural analysis of the carbon dots revealed that both MC-CDs and carbon dots formed using only Cur as a raw material (Cur-CDs) are rich in the characteristic D band (1360 cm⁻¹), representing a defect-like graphene core. -1 ) and G-band (1580cm) -1 The D peak represents defects or amorphous structures in carbon materials, primarily originating from sp. 3 Symmetry breaking of hybridized carbon atoms. The G peak represents the graphitized structure, originating from sp... 2In-plane stretching vibrations of hybrid carbon atoms. Calculations of the ID / IG values ​​revealed that Mn doping did not affect the formation of abundant defects on the carbon dot surface. The crystal structure of MC-CDs was characterized by XRD. The results showed a significant broad diffraction peak at 2θ = 25°, corresponding to the (002) crystal plane of graphitized carbon. Furthermore, a weak shoulder peak (d ≈ 0.21 nm) corresponding to the (100) crystal plane was observed near 41°, consistent with the lattice spacing in TEM. The presence of these characteristic diffraction peaks strongly confirms that the prepared carbon dots possess a typical graphitized carbon core structure (…). Figure 3 b). FTIR spectroscopy was used to characterize the surface functional groups and chemical bonds of MC-CDs. For example... Figure 3 As shown in c, at 3000 cm -1 Up to 3700 cm -1 The broad absorption characteristics within the range are attributed to the stretching vibrations of hydrophilic groups such as hydroxyl (-OH) and amino (-NH2) groups on the sample surface. At 1585.3 cm⁻¹... -1 1369.3 cm -1 The infrared absorption peaks at these locations correspond to -C=O / C=C and -OCH3, respectively, indicating that the carbon dot structure still contains a large number of Cur molecular functional groups, which provides a basis for MC-CDs to exert their antioxidant capacity in the later stages.

[0056] The elemental composition and chemical valence states of MC-CDs were systematically characterized using XPS. For example... Figure 4 As shown in Figure a, the full spectrum scan exhibits significant characteristic peaks at 285 eV, 401.2 eV, and 533 eV, which are attributed to the C 1s, N 1s, and O 1s energy levels, respectively. Due to the introduction of a metal precursor during synthesis, a characteristic peak of Mn 2p is clearly visible at 643 eV in the full spectrum, preliminarily confirming that manganese has been successfully integrated into the carbon dot structure. To further reveal the valence state distribution of manganese within the material, a high-resolution energy dispersive spectroscopy (HSDES) analysis was conducted. The HSDES of Mn 2p, after curve fitting, shows two distinct spin-orbit coupling double peaks, with characteristic peaks at 638.4 eV and 650.2 eV attributed to Mn 2p, respectively. 2+ 2p 3 / 2 and 2p 1 / 2 The energy levels, with peaks at 639.9 eV and 651.5 eV corresponding to Mn. 3+ 2p 3 / 2 and 2p 1 / 2 Energy levels. Experimental results show that multiple valence states of Mn elements are generated during carbon dot formation. This mixed valence state redox couple strongly supports its electron transfer capability in enzyme-like catalytic cycles. Figure 4 b). Figure 4The high-resolution C 1s spectrum in C1 shows three fitted peaks at 284.8 eV, 286.2 eV, and 287.8 eV, attributed to CC / C=C, CO / N, and C=O, respectively. This indicates that the characteristic groups of curcumin were retained during carbon point formation. Figure 4 The high-resolution O 1s spectrum in d shows Mn-O bonds at 529 eV, indicating successful integration of Mn elements and carbon dot framework.

[0057] Example 3: Antioxidant and Enzyme-like Activities of MC-CDs

[0058] In previous studies, we found that the prepared MC-CDs retained the characteristic functional groups of curcumin, potentially endowing them with strong free radical scavenging and antioxidant properties similar to curcumin. This invention first evaluated the antioxidant activity of MC-CDs using the ABTS method. ABTS can be oxidized to stable cationic free radicals in the presence of potassium persulfate, producing a characteristic absorption at 734 nm. For example... Figure 5 As shown in Figure a, MC-CDs exhibit excellent ABTS radical scavenging ability. At a concentration of 100 μg / mL, the scavenging rate reaches as high as 91%. Further analysis was performed using the DPPH method. DPPH is a stable free radical containing a single electron. Due to the presence of three benzene rings in its molecular structure, it can exist stably in single-electron form through conjugation stabilization and steric hindrance, and it has a characteristic absorption peak at 517 nm. The antioxidant performance of the material can be evaluated by detecting changes in this absorption peak. Figure 5 As shown in b, the DPPH scavenging effect of MC-CDs is concentration-dependent, reaching a scavenging rate of 89.8% at a concentration of 50 μg / mL. Furthermore, this invention also evaluated the scavenging effect of MC-CDs on ·OH. The experiment used the TMB colorimetric method, where ·OH is generated through the Fenton reaction between ferrous ions and hydrogen peroxide. TMB is oxidized by ·OH to form a blue product, which exhibits significant absorption at 652 nm. After the addition of an antioxidant, it competitively consumes ·OH to inhibit the oxidation of TMB, thereby reducing the absorbance at 652 nm. Figure 5 As shown in Figure c, MC-CDs at a concentration of 100 μg / mL achieved a scavenging rate of 85.5% for ·OH. Furthermore, electron spin resonance (ESR) characterization further confirmed the scavenging effect of MC-CDs on free radicals. Figure 5d). DMPO was used as a spin trapping agent to detect ·OH in the system. The results showed that the intensity of characteristic signals representing free radicals was significantly reduced after the addition of MC-CDs, demonstrating their excellent free radical scavenging ability at the electronic level. In summary, MC-CDs exhibit good broad-spectrum antioxidant activity, which further proves the feasibility of our design strategy for synthesizing antioxidant carbon dots based on curcumin as a carbon source.

[0059] Studies have shown that manganese is an excellent redox metal, and in MC-CDs it mainly exists as Mn. 2+ Mn 3+ Coexistence. This multivalent state characteristic constitutes an efficient electron transport chain, thus potentially endowing it with excellent enzyme-like activity. Among them, SOD enzymes can catalyze O2. •- A disproportionation reaction occurs, producing H₂O₂ and O₂, which play a central role in the body's defense against oxidative damage. Here, the SOD enzyme activity of MC-CDs was tested using a commercial WST kit, which utilizes the reaction between xanthine and xanthine oxidase to generate O₂. •- The reaction with WST produces formazan, which has strong absorption at 450 nm. Materials with SOD enzyme activity can inhibit the formation of formazan, thus accurately determining the SOD enzyme activity in the sample. For example... Figure 6 As shown in figure a, MC-CDs exhibit concentration-dependent SOD-like activity; at a concentration of 50 μg / mL, their SOD-like activity is 62 ± 2.9%. Possibly due to the influence of substrate concentration, the SOD-like activity of MC-CDs did not increase proportionally with concentration. ESR characterization ( Figure 6 b) This further confirms the effect of MC-CDs on O2. •- The scavenging effect was observed. DMPO was used as a spin trapping agent in the experiment to scavenge O2 in the system. •- The test was conducted. The results showed that adding MC-CDs represented O2. •- The characteristic signal intensity decreased significantly, strongly demonstrating its superior O2 performance at the electronic level. •- Clearance capability.

[0060] Furthermore, we employed a commercially available H2O2 detection method to test the CAT-like enzyme activity of MC-CDs. This assay utilizes the reaction of residual H2O2 in the system with the chromogenic substrate (TMB), generating a chromogenic product with characteristic absorption at 652 nm. Materials exhibiting CAT-like enzyme activity can effectively catalyze the decomposition of H2O2 in the system, inhibiting the formation of the chromogenic product by reducing the concentration of H2O2 within the reaction system. Therefore, by monitoring the decrease in absorbance after the reaction, the CAT-like enzyme activity of the sample can be accurately determined and evaluated. Figure 6c shows that MC-CDs exhibit concentration-dependent CAT enzyme activity; at a concentration of 50 μg / mL, their CAT enzyme-like activity is 71.2 ± 3.8%. Further, kinetic analysis of the scavenging H2O2 by MC-CDs was conducted... Figure 6 d) also shows that the addition of MC-CDs to a system containing H2O2 can continuously generate O2, further demonstrating its CAT-like catalytic ability. H2O2 and O2 are easily generated in pathological environments such as inflammation. •- Toxic free radicals can be eliminated, but based on the enzyme-like cascade catalysis of MC-CDs, they can be converted into harmless H2O and O2. This not only effectively removes ROS but also releases oxygen in situ, alleviating the hypoxic environment.

[0061] Example 4: Preparation and rheological, transmittance, and ocular retention characterization of CG@MC hydrogel eye drops.

[0062] While collagen hydrogels prepared by thermal assembly typically possess good mechanical properties, they often suffer from physical defects such as low optical transparency and poor injectability. This is mainly because, under suitable pH and temperature conditions, collagen molecules undergo rapid and disordered self-assembly, forming large fiber bundles and dense network structures. This microscopic topology leads to a strong light scattering effect, resulting in an opaque appearance. Our research found that pre-introducing a small amount of glycerol before the thermal assembly process can effectively interfere with the hydrogen bonding interactions between collagen molecules using its abundant hydroxyl groups. This competitive intermolecular binding significantly reduces the fiber diameter and aggregate size, making them much smaller than the wavelength of visible light. This ensures effective self-assembly of collagen while maintaining its biological structural integrity, while simultaneously endowing the hydrogel with high optical transparency and superior rheological properties.

[0063] This invention names the hydrogel eye drops prepared by adding glycerin as CG, and the composite system after introducing functional nanomaterials MC-CDs as CG@MC (prepared in Example 1). Figure 7 As shown, CG@MC exhibits excellent physical properties, allowing it to be smoothly extruded from the dropper bottle while maintaining extremely high transparency.

[0064] As a functionalized hydrogel eye drop used for corneal damage repair, it must possess excellent rheological properties to maintain the physiological curvature of the cornea and effectively buffer the periodic shear challenges generated during frequent blinking, within the complex physiological environment of the ocular surface. Figure 8 As shown in Figure a, CG hydrogel exhibits significant shear thinning within a shear rate range of 1-100 1 / s, and compared to Col hydrogel, it has lower viscosity, which is more conducive to achieving injectability. In frequency scanning (… Figure 8In (b), Col hydrogel exhibits a larger modulus, but this type of moldable hydrogel often causes eye discomfort when used in the eye. CG hydrogel with added glycerol shows a significant decrease in modulus compared to pure collagen hydrogel. This phenomenon is mainly attributed to the introduction of glycerol molecules regulating the self-assembly behavior of collagen fibers, forming a more flexible cross-linked network. Within the angular frequency scanning range of 0.1–100 rad / s, the storage modulus (G') of CG hydrogel is consistently significantly higher than the loss modulus (G''), and both exhibit good frequency independence. This result strongly demonstrates that CG hydrogel maintains stable gel properties over a wide frequency range, rather than a viscous fluid state. To further understand the ability of CG hydrogel to adapt to the dynamic blinking environment of the ocular surface, we designed a shear-creep test (…). Figure 8 c) The results showed that under a shear stress of 0.4 s, the CG hydrogel rapidly underwent strain, and the strain decreased rapidly after the shear stress was removed. This process demonstrates that the CG hydrogel has excellent dynamic environmental adaptability and is suitable for use as an eye drop with excellent biological activity.

[0065] As a hydrogel eye drop for ophthalmic use, excellent optical transparency is a necessary prerequisite to ensure effective tissue repair and functional reconstruction without interfering with the corneal "optical window" function. Figure 9 The results showed that the thermally assembled pure Col hydrogel exhibited low transmittance, visually hindering the observation of the background floral pattern. Transmittance testing also revealed poor transmittance at low wavelengths, with only 70% at 500 nm. In contrast, the CG hydrogel clearly displayed the background floral pattern, maintaining a high transmittance of 90% across the 250-800 nm wavelength range. To further explore the mechanism behind the transmittance changes, we used molecular simulations to investigate the mechanism of glycerol treatment of collagen gels, such as... Figure 9 As shown in b, after glycerol treatment, the molecular conformation of collagen molecules changes. The originally assembled, robust collagen fibers gradually separate into finer collagen fiber bundles. The reorganization of hydrogen bonds can be clearly observed in the magnified image. Further quantification of data ( Figure 9 (c) and (d) it was found that the number of hydrogen bonds formed between collagen molecules and glycerol increased rapidly with changes in spatial position, and then fluctuated within a certain range. The number of hydrogen bonds between collagen molecules was less than the number of hydrogen bonds between protein molecules in aqueous solution. These results indicate that the addition of glycerol molecules forms stronger bonds on the collagen molecule surface, hindering the formation of hydrogen bonds between collagen bundles. These results demonstrate that the introduction of glycerol molecules modulates the self-assembly behavior of collagen fibers, limiting the excessive aggregation of large fiber bundles by interfering with the hydrogen bonding interactions between collagen molecules, thereby endowing them with excellent rheology and light transmittance.

[0066] Traditional eye drops currently used in clinical applications generally face bottlenecks such as insufficient retention time on the ocular surface, rapid clearance by tears, and low bioavailability. To improve therapeutic efficacy, developing advanced drug delivery systems with long-lasting retention capabilities is crucial. To more intuitively and accurately evaluate the ocular surface retention characteristics of gels, this invention utilizes OCT technology for real-time visualization monitoring of gel distribution behavior in rat eyes. Figure 10 As shown in Figure a, at the initial stage of drug administration (0 min), both groups of rats had a distinct material layer covering their corneal surfaces. However, in the MC-CDs-only group, the material layer thickness rapidly decreased within 5 min as the rats blinked and moved, accumulating at the corners of the eyes. By 20 min, there was virtually no material residue left on the corneal surface, indicating that the small molecule solution was easily lost due to tear circulation and gravity.

[0067] In contrast, the CG@MC hydrogel group exhibited a significant retention advantage. At 5 and 20 minutes, a uniform and clear gel coating remained on the anterior corneal surface; even 40 minutes after drug administration, gel signals were still observable on OCT images. This result directly confirms that the CG hydrogel carrier can effectively delay drug loss, providing a lasting window of protection and treatment for damaged corneas. To further visually verify the retention characteristics of CG@MC, this invention introduced sodium fluorescein as a tracer, and the fluorescence distribution of the material was observed under cobalt blue light irradiation. Figure 10 As shown in b, the fluorescence signal of the MC-CDs solution group decayed drastically, with only extremely weak fluorescence visible at the corneal edge after 40 min. In contrast, the CG@MC group exhibited strong green fluorescence throughout the entire monitoring period, and strong fluorescence could still be observed after 40 min.

[0068] Combining OCT real-time monitoring and fluorescence tracking analysis, the CG@MC carrier exhibited significantly better retention on the corneal surface than the MC-CDs solution alone, with a stable material coating observed even 40 minutes after administration. This significant retention advantage is primarily attributed to the excellent rheological properties of CG hydrogel. Its superior shear-thinning behavior effectively resists natural tear erosion and maintains structural integrity during the blink cycle, thereby significantly enhancing the bioavailability of MC-CDs and providing robust physical support and microenvironmental protection for long-term corneal repair.

[0069] Example 5: Characterization of biocompatibility, antioxidant properties, and anti-inflammatory properties

[0070] To evaluate the safety of each material group in biological applications, this invention used HCECs as model cells and examined the cell compatibility of the Control, CG-Gel, MC-CDs, and CG@MC groups. CG-Gel was a collagen hydrogel prepared by adding glycerol to a collagen solution, without the addition of MC-CDs. Figure 11 As shown, the live / dead cell staining results demonstrated the good biocompatibility of each group of materials. All treatment groups exhibited uniform cell density distribution and strong green fluorescence, with almost no red fluorescence signal observed, indicating that CG-Gel, MC-CDs, and the CG@MC composite system had no adverse effects on cell growth. Furthermore, the MTT proliferation assay results ( Figure 12 This further validated the conclusions drawn from the live-dead staining experiment. On day 3 of culture, there was no significant difference in cell viability between the experimental groups and the control group. This result lays the foundation for its subsequent application in corneal tissue repair.

[0071] Because MC-CDs contain antioxidant and enzyme-like activities, we examined their antioxidant properties at the cellular level using DPPH free radical scavenging assays and dissolved oxygen assays with oxygen indicators. Figure 13 As shown in a, we selected HCECs as model cells. When adding the materials, we added 50 μM H2O2 to create an oxidative environment. After incubation for 12 h, we stained and observed the changes in cell fluorescence.

[0072] First, intracellular ROS levels were characterized using DCFH-DA staining. (See illustration) Figure 13 As shown in b, under H2O2 induction, cells in the Control and CG-Gel groups produced a large amount of ROS. These cells exhibited significant green fluorescence under the action of DCFH-DA dye. In contrast, the MC-CDs and CG@MC groups only showed blue fluorescence representing the cell nucleus, indicating that almost all ROS induced by H2O2 was eliminated. Figure 13 The quantitative statistical results of the green fluorescence intensity in c also confirm the above conclusion. Due to the presence of antioxidant MC-CDs, CG@MC hydrogel can protect cells from ROS damage in an oxidative environment, which is of great significance for corneal damage repair under inflammatory conditions.

[0073] Because MC-CDs possess enzyme-like activity capable of converting superoxide radicals and hydrogen peroxide into oxygen, we further evaluated their oxygen-releasing capacity in oxidizing environments using the oxygen indicator [Ru(dpp)3]Cl2. In the absence of oxygen or under low oxygen concentrations, it emits a strong red fluorescence. Its fluorescence is quenched by oxygen as the oxygen concentration increases. Figure 13As shown in figures d and e, significant red fluorescence was observed in both the Control and CG-Gel groups, and statistical data showed no significant difference in fluorescence intensity between the two, indicating that the pure hydrogel did not improve the oxidative environment. However, no red fluorescence was observed in the MC-CDs and CG@MC groups, indicating oxygen quenching. Compared to the MC-CDs group, the oxygen quenching effect was more pronounced in the CG@MC group, which may be due to the increased dispersion of carbon dots after the introduction of the hydrogel, preventing carbon dot aggregation and thus affecting cell viability. These results collectively demonstrate the excellent cell protection and oxygen production capabilities of the CG@MC hydrogel.

[0074] CG@MC hydrogel possesses significant immunomodulatory functions, driving the transformation of pro-inflammatory M1 macrophages into anti-inflammatory M2 macrophages, thereby remodeling the inflammatory microenvironment in the corneal injury area. To systematically evaluate the ability of CG@MC hydrogel to regulate macrophage polarization, this invention utilized lipopolysaccharide (LPS) to induce M1 polarization in RAW 264.7 macrophages, and then co-incubated them with various other materials for 24 hours. Figure 14 a).

[0075] Immunofluorescence image results showed ( Figure 14 (b) RAW 264.7 cells stimulated with LPS showed high expression of the pro-inflammatory cytokine IL-6, confirming that macrophages had successfully polarized from a resting state to a pro-inflammatory M1 phenotype. However, the expression of IL-6 in RAW 264.7 cells was significantly reduced after treatment with MC-CDs or CG@MC hydrogel. This is mainly attributed to the excellent enzyme-like activity and antioxidant effects of MC-CDs, which effectively cleared excess ROS accumulated intracellularly and blocked the activation of pro-inflammatory signaling pathways. Furthermore, CG@MC hydrogel also promoted the expression of the anti-inflammatory cytokine IL-10. Figure 14 (d) indicates that the composite system can efficiently induce the transformation of M1 macrophages to M2.

[0076] Immunofluorescence quantitative analysis results ( Figure 14 (c, e) Consistent with morphological observations, the CG@MC group exhibited the lowest IL-6 expression level and the highest IL-10 level. In conclusion, CG@MC hydrogel loaded with MC-CDs can significantly inhibit inflammatory damage in the corneal injury area by precisely regulating the polarization state of macrophages, creating an ideal immune microenvironment for the repair and regeneration of damaged tissues.

[0077] Example 6: Application of CG@MC hydrogel in corneal alkali burns

[0078] Corneal epithelialization and stromal regeneration

[0079] To thoroughly evaluate the repair effect of CG@MC hydrogel eye drops on corneal alkali burns, a standard corneal alkali burn model was established using SD rats. First, the ocular surface of the rats was observed using a slit-lamp microscope. Figure 15 (a, b) Results showed that on the day of modeling (Day 0), the damaged cornea presented a typical grayish-white translucent appearance. After staining with sodium fluorescein, the cornea exhibited a strong green fluorescence signal under cobalt blue light, indicating that the corneal epithelium had completely sloughed off. By the 4th postoperative day, all groups of corneas showed varying degrees of stromal edema and opacity. Although the green fluorescence area in the Control group and the CG-Gel group decreased, corneal edema remained severe and white opacity intensified. In contrast, the CG@MC group showed excellent early repair, with significantly improved corneal transmittance and a markedly accelerated epithelialization process.

[0080] At 21 days post-surgery, significant differences in repair outcomes were observed among the groups. The corneas of the Control and CG-Gel groups ultimately developed into severe scar tissue, accompanied by corneal neovascularization (CNV) invading the central region. Figure 15 c). Although the pure MC-CDs eye drops group promoted epithelial regeneration to some extent in the early stages, the cornea remained cloudy in the later stages, accompanied by significant CNV formation. The main reason is that, in the complex ocular surface environment, the effective drug concentration of liquid eye drops cannot be maintained for a long time due to frequent blinking and tear rinsing.

[0081] In the CG@MC hydrogel group, the cornea essentially returned to a physiologically transparent state 21 days post-surgery, with no obvious scarring. The introduction of the hydrogel carrier not only significantly enhanced the material's retention on the ocular surface but also effectively curbed corneal edema and confined neovascularization to the corneal limbus region through its continuously released antioxidant and anti-inflammatory components. Epithelialization statistics (…) Figure 15 d) Further quantitative confirmation of the superior efficacy of CG@MC showed that the rats in this group achieved rapid corneal epithelial reconstruction as early as 4 days after surgery. However, due to persistent inflammatory interference, the other three groups still had a small amount of epithelial defects 21 days after surgery.

[0082] We further used OCT to non-invasively monitor the corneal stromal structure during the corneal repair process in each group. For example... Figure 16As shown in Figure a, after alkali burns, the corneal stroma rapidly thickened due to severe inflammatory edema, exhibiting typical pathological thickening characteristics. By postoperative day 4, due to the continued inflammatory response, the stromal edema in the Control and CG-Gel groups had not significantly subsided. In contrast, the edema in the MC-CDs and CG@MC groups, which possess multi-enzyme activity and in-situ oxygen production capabilities, had begun to lessen. At postoperative day 21, the corneal thickness in the Control group showed an abnormal and continuous increase, primarily attributed to the long-term stimulation of inflammatory factors inducing excessive activation and proliferation of fibroblasts, leading to severe fibrosis of the corneal stroma and the formation of opaque scar tissue.

[0083] In the CG@MC group, thanks to the material's long-lasting antioxidant and anti-inflammatory effects, OCT images showed that the outermost layer of the cornea had been reconstructed with a continuous and smooth high-reflectivity bright line, indicating complete coverage of the corneal epithelium and restoration of the stromal layer. (Statistical data) Figure 16 (b) Further quantitative confirmation showed that, 21 days post-surgery, the corneal thickness in the CG@MC group had returned to physiological levels, with no significant difference compared to the normal group. In contrast, although the MC-CDs group alone alleviated stromal edema to some extent, due to the limited effective action time of the liquid preparation on the ocular surface, a certain degree of stromal opacity and scarring still occurred in the later stages.

[0084] We prepared MC-CDs nanozymes with dual-valence manganese doping and multiple enzyme-like activities using a solvothermal method via the reaction of manganese ions with curcumin. This demonstrated that the abundant functional groups and mixed-valence manganese (Mn) on the surface of the MC-CDs nanozymes... 2+ / Mn 3+ These factors combined to endow the nanomaterial with broad-spectrum antioxidant activity and multi-enzyme catalytic cascade function. The retention of curcumin functional groups confers its potent superoxide dismutase (SOD) and catalase (CAT) activities, enabling it to scavenge •OH and ABTS. +Free radicals such as DPPH are eliminated. Through a cascade enzyme catalysis, ROS in the pathological microenvironment are converted into O2, alleviating hypoxia, downregulating the HIF-1α / VEGF pathway at its source, inhibiting angiogenesis, and calming the inflammatory response. Furthermore, an appropriate amount of glycerol was introduced during the preparation of the collagen gel. Utilizing its competitive hydrogen bond formation with collagen side chains, it effectively reduced the fiber diameter and aggregation size. This microstructural reconstruction not only endowed the collagen hydrogel with high light transmittance (>95%) but also gave it significant shear-thinning rheological properties, enabling the system to be used as a high-performance hydrogel eye drop. While maintaining convenient eye drop administration, it significantly enhanced the material's residence time on the ocular surface (>40 min). By incorporating MC-CDs nanozymes into collagen-based hydrogels (CG) with glycerol-regulated self-assembly behavior, CG@MC hydrogel eye drops with high transparency and long-lasting ocular surface retention were successfully prepared. These drops exhibit good cell compatibility and, under simulated pathological conditions, efficiently scavenge ROS and release oxygen in situ through an enzyme-like cascade reaction, effectively protecting corneal epithelial cells from oxidative stress-induced damage and apoptosis. Simultaneously, this system promotes macrophage polarization towards the anti-inflammatory M2 type by downregulating IL-6 and upregulating IL-10 and Arg-1 expression, achieving remodeling of the local immune microenvironment. In in vivo experiments of alkali-induced corneal burns in rats, this composite system synergistically improved early acute inflammatory damage and late-stage hypoxia, promoted rapid corneal epithelial reconstruction, and effectively blocked α-SMA-mediated scar hyperplasia and CD31-mediated neovascularization, ultimately restoring corneal thickness and transparency to physiological levels. This innovative hydrogel product holds promise as a clinical material for treating corneal alkali burns and is of great significance for the clinical translation of corneal regenerative medicine.

Claims

1. A manganese-curcumin carbon dot MC-CDs, characterized in that... It is prepared by using curcumin as a carbon source and manganese ions as a metal dopant raw material, and adopting an alkaline-assisted hydrothermal carbonization method to prepare manganese-curcumin carbon dots MC-CDs, wherein the mass ratio of manganese ions to curcumin is 8-9:

100.

2. The manganese-curcumin carbon dots MC-CDs according to claim 1, characterized in that: The mass ratio of manganese ions to curcumin is 8.73:

100.

3. The manganese-curcumin carbon dots MC-CDs according to claim 1 or 2, characterized in that: The manganese ions are divalent or trivalent manganese ions, including manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; the particle size range of the manganese-curcumin carbon dots MC-CDs is 2.6-5.9 nm.

4. The manganese-curcumin carbon dots MC-CDs according to claim 1 or 2, characterized in that: The raw materials also include one of formamide, methanol, acetamide, and ethylene glycol, with a mass-to-volume ratio of 1:2 to curcumin.

5. The method for preparing manganese-curcumin carbon dots (MC-CDs) according to any one of claims 1-4, characterized in that: It includes the following steps: a. Add curcumin to formamide and add NaOH as a solubilizer to prepare a precursor solution; b. Pretreatment: Under constant speed magnetic stirring, manganese chloride was slowly introduced into the precursor solution and homogeneous stirring was maintained for 30 min. c. Hydrothermal carbonization: The mixture is transferred to a high-pressure reactor lined with polytetrafluoroethylene and reacted at 180°C for 4 hours. d. Purification: After the reaction system cooled naturally to room temperature, the product was filtered through a 0.22 μm microporous membrane and then dialyzed with a molecular weight cutoff of 1000 Da. The dialysis process was carried out in ultrapure water for 72 h, with water changed every 6 h to ensure that impurities were completely removed. e. Product collection: The dark green solid powder is collected by vacuum freeze-drying technology and sealed and stored in a desiccator.

6. The use of the manganese-curcumin carbon dots MC-CDs according to any one of claims 1-4 in the preparation of ophthalmic drugs for treating corneal alkali burns; or, the use of the manganese-curcumin carbon dots MC-CDs in the preparation of topical drugs with antioxidant and anti-inflammatory effects.

7. An ophthalmic pharmaceutical composition for treating corneal alkali burns, characterized in that: It is an ophthalmic preparation made from manganese-curcumin carbon dots MC-CDs as described in any one of claims 1-4 and collagen as raw materials. The mass ratio of manganese-curcumin carbon dots MC-CDs to collagen is 1:2 to 1:5, and the optimal mass ratio is 1:

4.

8. The ophthalmic pharmaceutical composition for treating corneal alkali burns according to claim 7, characterized in that: The ophthalmic preparations mentioned are eye drops, eye ointments, and ophthalmic gels.

9. A method for preparing the ophthalmic pharmaceutical composition for treating corneal alkali burns according to claim 7 or 8, characterized in that... The preparation method of the eye drops includes the following steps: a) Dissolve collagen in glacial acetic acid solution to prepare a collagen solution, and add an equal volume of PBS solution; b. Dissolve manganese-curcumin carbon dots (MC-CDs) in 1% glycerol; c. Add the MC-CDs / glycerol solution prepared in step a to the collagen / acetic acid solution prepared in step a, adjust the pH to 7.4 with sodium hydroxide, and place at 37°C to obtain the eye drops of the present invention.

10. Use of the ophthalmic pharmaceutical composition of claim 7 or 8 in the preparation of a medicament for treating corneal alkali burns.

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