Copper ion-sargentgloryvine stem carbon quantum dot nano-enzyme as well as preparation method and application thereof

Abstract

The invention discloses a copper ion-sargentgloryvine stem carbon quantum dot nano-enzyme as well as a preparation method and application thereof, and relates to the technical field of biological medicines. The preparation method comprises the following steps: mixing copper ions, sargentgloryvine stem and water, performing hydrothermal reaction, and centrifuging to obtain supernate; and carrying out filtration treatment, dialysis treatment and drying treatment on the supernate to obtain the copper ion-sargentgloryvine stem carbon quantum dot nano-enzyme. The nano-enzyme can obviously improve corneal injury caused by xerophthalmia, prolong tear film rupture time, increase tear secretion amount and obviously reduce the ROS level of corneal tissue, is high in ocular surface retention capacity and has no toxicity to important organs such as heart, liver and spleen. Compared with positive control drug sodium hyaluronate eye drops, the nano-enzyme has the advantages of inflammation resistance, oxidation resistance and biological safety, provides a novel and efficient treatment strategy for xerophthalmia treatment, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme, its preparation method, and its application. Background Technology

[0002] Dry eye syndrome, also known as keratoconjunctivitis sicca (DED), is a common and complex ophthalmic disease caused by insufficient tear secretion, excessive evaporation, or abnormal tear composition. It can cause eye discomfort and visual impairment. During an attack, patients experience significant dryness, fatigue, and a foreign body sensation in the eyes. In severe cases, patients may experience eye pain, photophobia, burning, soreness, and redness, greatly interfering with daily activities. If left untreated, it can lead to secondary infections, corneal ulcers or perforation, and in more severe cases, blindness. With technological advancements and the exponential increase in electronic device use, coupled with environmental degradation and widespread light pollution, the prevalence of DED has also seen explosive growth. Currently, there is no cure for DED; treatment strategies primarily focus on symptom relief. Traditional medications include artificial tears that mimic the tear environment and antibiotic eye drops, but these have drawbacks such as short retention time and significant side effects. Prolonged use can also lead to the accumulation and metabolism of preservatives and excipients, further burdening the ocular surface and worsening the condition. Currently, the recognized causes of diabetic retinopathy (DED) include ocular inflammation and tear film hyperosmolarity. For example, increased ocular surface osmotic pressure, decreased tear secretion, and ocular surface dryness trigger inflammatory mechanisms, leading to the release of pro-inflammatory cytokines, matrix metalloproteinases, and chemokines. Inflammation results in elevated levels of reactive oxygen species (ROS), exacerbating DED. Nanozyme strategies targeting ROS level regulation have become a popular research strategy to replace traditional treatments. Summary of the Invention

[0003] The purpose of this invention is to provide a copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme, its preparation method, and its applications, to solve the problems existing in the prior art. This copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme can effectively improve dry eye syndrome, providing a novel and efficient treatment strategy for dry eye syndrome, with broad application prospects.

[0004] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for preparing copper ion-Sargentodoxa cuneata carbon quantum dot nanozymes, comprising the following steps: Copper ions, *Sargentodoxa cuneata*, and water were mixed and subjected to a hydrothermal reaction. The supernatant was then obtained by centrifugation. After filtration, dialysis and drying of the supernatant, the copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme was obtained.

[0005] Furthermore, the temperature of the hydrothermal reaction is 180°C.

[0006] Furthermore, the hydrothermal reaction time is 8 hours.

[0007] Furthermore, the mass ratio of the copper ions to the *Sargentodoxa cuneata* is 1:10.

[0008] Furthermore, the filtration process uses a 0.22 μm filter membrane.

[0009] Furthermore, the dialysis treatment uses a 400 Da dialysis bag.

[0010] The present invention also provides a copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme prepared according to the above preparation method.

[0011] The present invention also provides the application of the above-mentioned copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme in the preparation of drugs for treating dry eye syndrome.

[0012] The present invention also provides a drug for treating dry eye syndrome, the active ingredient of which includes the above-mentioned copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme.

[0013] Furthermore, the drug also includes pharmaceutically acceptable excipients.

[0014] The present invention discloses the following technical effects: This invention develops a copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme (Cu@CSG@CDS), which achieves stable binding of copper ions and Sargentodoxa cuneata carbon quantum dots through a hydrothermal synthesis process. Characterization results confirm that it possesses good physical stability, a near-spherical morphology, and specific optical and crystal properties. In vitro experiments show that it has excellent biocompatibility and can be efficiently taken up by HCE-T cells and accumulate in a time-dependent manner. In a hyperosmolar injury cell model, this nanozyme can significantly improve cell survival rate, effectively scavenge intracellular reactive oxygen species (ROS), inhibit mitochondrial membrane potential damage, and reduce cell apoptosis, with effects superior to ungrafted Sargentodoxa cuneata carbon quantum dots (CSG@CDS). In in vivo experiments, it can significantly improve corneal damage caused by dry eye syndrome, prolong tear film breakup time, increase tear secretion, significantly reduce corneal tissue ROS levels, and has strong ocular surface retention capacity, with no toxicity to vital organs such as the heart, liver, and spleen. Compared to the positive control drug sodium hyaluronate eye drops, this nanozyme has the advantages of anti-inflammatory, antioxidant and biocompatibility, providing a new and efficient treatment strategy for dry eye syndrome, with broad application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 Potential diagrams for CSG@CDS and Cu@CSG@CDS; Figure 2 TEM images of CSG@CDS and Cu@CSG@CDS; Figure 3 UV-vis plots for CSG@CDS and Cu@CSG@CDS; Figure 4 FTIR plots of CSG@CDS and Cu@CSG@CDS; Figure 5 XPS plots for CSG@CDS and Cu@CSG@CDS; Figure 6 XRD patterns for CSG@CDS and Cu@CSG@CDS; Figure 7 The graph shows the cytotoxicity test results of RAW264.7 under 24 h conditions in each drug administration group; Figure 8 The graph shows the cytotoxicity test results of HCE-T in each drug administration group under 24 h conditions; Figure 9 Laser confocal images (Bar=20 μm) of Cu@CSG@CDS and HCE-T cells after co-incubation for 1, 3, and 5 h (A) and semi-quantitative analysis of intracellular FITC content (B). Figure 10 The graph shows the cytotoxicity test results of different concentrations of NaCl. Figure 11 Statistical graph showing the cell viability of HS cell models under different concentrations of CSG@CDS (A) and Cu@CSG@CDS (B); Figure 12 The graph shows the cytotoxicity test results under different concentrations of Cu@CSG@CDS and CSG@CDS. Figure 13 Inverted fluorescence image of HCE-T cells co-stained with Calcein-AM / PI (Bar=100 μm). Figure 14 Fluorescent inverted image (Bar=100 μm) for DHE staining to detect intracellular ROS levels in HCE-T cells (A) and semi-quantitative analysis of intracellular ROS levels (B); Figure 15 Fluorescent images of mitochondrial membrane potential damage in HCE-T cells detected by JC-1 staining (Bar=100 μm) (A) and semi-quantitative analysis of intracellular JC-1 levels (B); Figure 16 Photographs of the eyeballs of mice in each group were taken using a cobalt blue light slit microscope; Figure 17 A statistical graph showing the fluorescein sodium staining score of mouse corneal epithelial tissue; Figure 18 A statistical graph of tear film breakup time in mice; Figure 19 A statistical graph showing the change in body weight of mice during the drug administration period; Figure 20 A statistical graph showing the amount of tear secretion in mice as measured by the Schirmer test. Figure 21 Fluorescence image of DHE-stained corneal tissue (Bar=100 µm). Figure 22 A statistical graph showing the fluorescence intensity of DHE in tissues for semi-quantitative analysis; Figure 23 Fluorescence images of FITC-Cu@CSG@CDS retention on the ocular surface in different groups; Figure 24 Statistical graph of in vivo fluorescence ROI values ​​for different groups; Figure 25 DAPI staining images of FITC-Cu@CSG@CDS retention at different time points (Bar=100 µm). Figure 26 This is a pathological image of corneal epithelial tissue stained with HE (Bar=50 µm). Figure 27 HE staining pathological images of important organs in mice after treatment (Bar=100 µm). Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0020] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0021] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0022] Example 1 1. Synthesis of Sargentodoxa cuneata nanozyme Weigh about 1 g of Sargentodoxa cuneata Chinese herbal medicine slices, ultrasonically clean them in a distilled water environment for 2 - 5 min, repeat the operation 2 - 3 times, and use sterile scissors to cut the Sargentodoxa cuneata Chinese herbal medicine slices into small pieces with uniform volume (about 0.5 cm × 0.5 cm). Add the medicinal material fragments to deionized water, transfer them to a high-pressure reaction kettle, keep the temperature at 180 °C and react for 8 h, then centrifuge (12000 r / min, 15 min), filter the supernatant through a filter membrane (0.22 μm), dialyze with deionized water for three days (400 Da dialysis bag, change deionized water every 8 h), and rotary evaporate and concentrate to obtain Sargentodoxa cuneata nanozyme (CSG@CDS).

[0023] 2. Synthesis of Cu-Sargentodoxa cuneata nanozyme After mixing Cu(II) and Sargentodoxa cuneata Chinese herbal medicine slices according to a mass ratio of 1:10, add deionized water, transfer them to a high-pressure reaction kettle, keep the temperature at 180 °C and react for 8 h, then centrifuge (12000 r / min, 15 min), filter the supernatant through a filter membrane (0.22 μm) to obtain a filtrate, and then dialyze with deionized water for three days (400 Da dialysis bag, change deionized water every 8 h). Take the retentate in the dialysis bag, and rotary evaporate and concentrate to obtain Cu-Sargentodoxa cuneata nanozyme (Cu@CSG@CDS).

[0024] 2. Characterization of nanozyme The potential diagrams of CSG@CDS and Cu@CSG@CDS are shown in Figure 1CSG@CDS exhibits negative electronegativity and good stability. The absolute value of the ζ-potential of Cu@CSG@CDS is increased, which indicates an increase in its physical stability.

[0025] TEM images of CSG@CDS and Cu@CSG@CDS are shown below. Figure 2 The CSG@CDS and Cu@CSG@CDS particles are spherical in shape, and their particle size results show a consistent trend.

[0026] A UV spectrophotometer was used to perform a full-wavelength UV scan analysis on CSG@CDS and Cu@CSG@CDS, and the absorbance in the 200-800 nm range was as follows: Figure 3 As shown, the maximum absorption wavelength of Cu@CSG@CDS is 277 nm, and the maximum absorption wavelength of CSG@CDS is 281 nm.

[0027] The FTIR plots of CSG@CDS and Cu@CSG@CDS are shown below. Figure 4 Compared to CSG@CDS, Cu@CSG@CDS performs better in the 2000-2500cm range. -1 The change in the absorption peak is caused by the stretching vibration of the CO-Cu bond, indicating that Cu@CSG@CDS synthesis was successful.

[0028] See the XPS charts for CSG@CDS and Cu@CSG@CDS. Figure 5 Both CSG@CDS and Cu@CSG@CDS contain characteristic peaks of C1s, N1s, and Ols, while Cu@CSG@CDS detects a Cu 2p characteristic peak at 980 eV.

[0029] The crystal structures of CSG@CDS and Cu@CSG@CDS were analyzed using XRD, such as... Figure 6 As shown, the horizontal axis represents the 2θ diffraction angle, and the vertical axis represents the intensity. Unlike CSG@CDS, Cu@CSG@CDS produces diffraction peaks at 2θ angles of 11.69°, 20.68°, 29.12°, and 31.1°, proving that Cu@CSG@CDS was successfully synthesized.

[0030] Example 2 1. Experimental Methods 1.1 Cytotoxicity test HCE-T cells and RAW 264.7 cells were resuspended at 10⁻⁶. 4Cells were seeded at a density of 100 μL per well into 96-well plates. The plates were gently shaken and allowed to settle to ensure even distribution. The plates were then incubated at 37°C with 5% CO2 for initial cell culture. After 24 h, cells were co-cultured with 0, 6.25, 12.5, 25, 50, and 100 μg / mL CSG@CDS and Cu@CSG@CDS for another 24 h. The effects of CSG@CDS and Cu@CSG@CDS on the proliferation of HCE-T and RAW 264.7 cells were then assessed using CCK-8 assay.

[0031] 1.2 Cell uptake assay Fluorescein isothiocyanate (FITC) and Cu@CSG@CDS were added to a 50% ethanol solution at a mass ratio of 1:10. After stirring magnetically for 24 h in the dark at room temperature, the mixture was centrifuged (12000 rpm, 15 min). The precipitate was washed three times with deionized water to obtain FITC-labeled Cu@CSG@CDS (FITC-Cu@CSG@CDS). HCE-T cells were attached to coverslips, and FITC-Cu@CSG@CDS at a concentration of 200 μg / mL was added to the cell supernatant. The cells were incubated for 1, 3, and 5 h, and gently washed three times with PBS. The cells were then fixed in 4% paraformaldehyde fixative for 15 min. The fixative was discarded, and the cells were washed three times again with PBS. The cells were then incubated with DAPI staining solution for 5 min at room temperature. After incubation, the cells were washed three times with PBS. The fluorescence intensity of the cells was observed and photographed using an inverted fluorescence microscope at 1, 3, and 5 h. The fluorescence images were quantified using ImageJ.

[0032] 1.3 Establishment of a hyperosmolar injury cell model High-concentration sodium chloride (NaCl) solution can establish a high osmolar (HS) injury model in HCE-T cells. The CCK-8 assay was used to detect the degree of damage to HCE-T cells caused by different concentrations of NaCl solution. After cell counting using a counting chamber, the cell suspension was seeded into 96-well plates (100 μL / well, cell density 1 × 10⁶ cells / well). 4 Cells were placed in a 37℃, 5% CO2 incubator for pre-culture. After 24 h, the cells were co-cultured with NaCl solutions of 0, 50, 100, 300, 500, and 800 mOsm / L for 24 h, and the cell viability was then detected by the CCK-8 assay.

[0033] 1.4 Treatment of hyperosmolar injury cell models with Cu@CSG@CDS and CSG@CDS The therapeutic effects of Cu@CSG@CDS and CSG@CDS on a HS-damaged cell model were detected using the CCK-8 assay. After cell counting using a counting chamber, HCE-T cell suspension was seeded into 96-well plates (100 μL / well, cell density 1×10⁻⁶ cells / well). 4 Cells were pre-cultured at 37℃ in a 5% CO2 incubator. After 24 h, the cells were co-cultured with 500 mOsm / L NaCl solution for 24 h to establish a HS injury model. The culture medium was then discarded, and the cells were co-cultured with 0, 25, 50, 100, and 200 μg / mL Cu@CSG@CDS and CSG@CDS solutions for 24 h. Cell viability was then assessed using the CCK-8 assay.

[0034] 1.5 Cell live / dead staining assay HCE-T cells were co-stained with Calcein-AM and PI to analyze the in vitro anti-inflammatory effect of HPB. The experiment consisted of four groups: Control group, HS group, 150 μg / mL CSG@CDS+HS group, and 150 μg / mL Cu@CSG@CDS+HS group. Cells were evenly seeded into six-well plates, with 2-3 mL of cell suspension (5-10 × 10⁶ cells / well) per well. 5 Cells were cultured in 500 mOsm / well solutions and allowed to adhere for 24 hours. Afterward, the culture medium was removed. Except for the control group, all other groups were incubated with serum-free culture medium containing 500 mOsm / L NaCl for 12 hours. After incubation, the NaCl-containing culture medium was discarded, and serum-free culture medium, 150 μg / mL CSG@CDS, and 150 μg / mL Cu@CSG@CDS were added sequentially. Cells were incubated for 12 hours, the culture medium was discarded, and the cells were washed once with PBS. The PBS solution was discarded, and the cells were fixed in 4% paraformaldehyde fixative for 15 minutes. The fixative was discarded, and the cells were washed three times again with PBS. 1 mL of 500 μM Calcein-AM and 100 μM PI dye was added, and incubation continued for 15 minutes. The cells were washed twice with PBS to remove unbound dye, and cell viability was observed using an inverted fluorescence microscope.

[0035] 1.6 Intracellular Reactive Oxygen Species (ROS) Scavenging Assay Intracellular ROS clearance was assessed using dihydroethidium (DHE). The experiment consisted of four groups: Control group, HS group, 150 μg / mL CSG@CDS+HS group, and 150 μg / mL Cu@CSG@CDS+HS group. HCE-T cells in the logarithmic growth phase were digested and evenly seeded into six-well plates, with 2-3 mL of cell suspension (5-10 × 10⁶ cells / well) per well. 5Cells were cultured in 6-well plates (number per well). After 24 h of cell adhesion, the cell culture medium was removed. Except for the control group, serum-free culture medium containing 500 mOsm / L NaCl was added to all other groups, and the plates were incubated for 12 h. After incubation, the NaCl-containing culture medium was discarded, and serum-free culture medium, 150 μg / mL CSG@CDS, and 150 μg / mL Cu@CSG@CDS were added sequentially. The plates were incubated for 12 h, and the culture medium was discarded. The cells were then washed once with PBS. The culture medium was diluted with DHE working solution (10 μm, 1 mL), and incubated at room temperature or 37 °C for 60 min. The cells were rinsed 1-2 times with PBS to thoroughly remove any DHE probes that had not penetrated the cells. The cells were fixed in 4% paraformaldehyde for 15 min, the fixative was discarded, and the cells were washed three times again with PBS. The samples were observed and analyzed using an inverted fluorescence microscope, and the fluorescence intensity was quantitatively assessed using ImageJ image analysis software.

[0036] 1.7 Measurement of mitochondrial membrane potential damage Mitochondrial membrane potential changes were detected using a mitochondrial membrane potential assay kit (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide, JC-1). The experiment consisted of four groups: Control group, HS group, 150 μg / mL CSG@CDS+HS group, and 150 μg / mL Cu@CSG@CDS+HS group. HCE-T cells in the logarithmic growth phase were digested and evenly seeded into six-well plates, with 2-3 mL of cell suspension (5-10 × 10⁶ cells / well) per well. 5 (cells / well) After 24 h of cell adhesion, the cell culture medium was removed. Except for the control group, all other groups were incubated with serum-free culture medium containing 500 mOsm / L NaCl for 12 h. After incubation, the NaCl-containing culture medium was discarded, and serum-free culture medium, 150 μg / mL CSG@CDS, and 150 μg / mL Cu@CSG@CDS were added sequentially. The cells were incubated in a cell culture incubator for 12 h. After discarding the culture medium, the cells were washed once with PBS solution. After discarding the culture medium, 1 mL of JC-1 dye was added, and the cells were incubated at 37℃ for 2 h. The cells were washed twice with PBS to remove unbound probes. The cells were fixed in 4% paraformaldehyde fixative, the fixative was discarded, and the cells were washed again with PBS solution. The damage to the mitochondrial membrane potential in the cells was observed under an inverted fluorescence microscope, and the fluorescence intensity was quantitatively assessed using ImageJ image analysis software.

[0037] 2. Experimental Results 2.1 RAW 264.7 Cytotoxicity Evaluation The cellular safety of CSG@CDS and Cu@CSG@CDS was evaluated using RAW 264.7 cells. Figure 7 As shown, RAW264.7 cells were co-cultured with different concentrations of CSG@CDS and Cu@CSG@CDS (0, 6.25, 12.5, 25, 50, 100 μg / mL) for 24 h. At a concentration of 100 μg / mL, the cell viability was still above 70%, and the survival rate of nanoparticles at each concentration gradient was similar to that of the control.

[0038] 2.2 Evaluation of HCE-T cytotoxicity The cellular safety of CSG@CDS and Cu@CSG@CDS was evaluated using HCE-T cells. For example... Figure 8 As shown, HCE-T cells were co-cultured with different concentrations of CSG@CDS and Cu@CSG@CDS (0, 6.25, 12.5, 25, 50, 100 μg / mL) for 24 h. At a concentration of 100 μg / mL, the cell viability was still above 70%.

[0039] 2.3 HCE-T cell uptake The in vitro uptake capacity and therapeutic effect of nanozymes were observed through cellular uptake assays. First, fluorescein isothiocyanate (FITC) was conjugated with Cu@CSG@CDS to obtain FITC-Cu@CSG@CDS. Then, HCE-T cells were co-incubated with 150.0 μg / mL FITC-Cu@CSG@CDS for 1, 3, and 5 h, respectively. Finally, the cells were observed using an inverted fluorescence microscope, and quantitative fluorescence was performed using ImageJ to evaluate the uptake effect of Cu@CSG@CDS by HCE-T cells. Figure 9 As shown in Figure A, Cu@CSG@CDS treatment for 1 h, 3 h, and 5 h resulted in gradually increasing green fluorescence of FITC within the cells, demonstrating that Cu@CSG@CDS can be effectively taken up by HCE-T cells. Figure 9 As shown in Figure B, the fluorescence intensity (MFI) of FITC-Cu@CSG@CDS continuously increases over time, exhibiting a time-dependent effect.

[0040] 2.4 Establishment of the HS cell injury model The CCK-8 assay was used to investigate the damage of different concentrations of NaCl solution to HCE-T cells. The success of the model was assessed by observing cell viability after 24 h of co-culturing with HCE-T cells. Figure 10As shown, a 50 mOsm / L NaCl solution resulted in a cell viability of 96.55% ± 0.25%, with no effect on cell viability. However, as the NaCl concentration increased, the cell viability gradually decreased. When the NaCl concentration reached 500 mOsm / L, the cell viability was only 55.06% ± 3.05%, indicating a significant decline. Therefore, a NaCl concentration of 500 mOsm / L was chosen for subsequent experiments.

[0041] 2.5 Therapeutic effects of Cu@CSG@CDS and CSG@CDS on HS cell models The CCK-8 assay was used to investigate the damage of different concentrations of NaCl solution to HCE-T cells. The success of the model was assessed by observing cell viability after 24 h of co-culturing with HCE-T cells. Figure 11 As shown, after the successful establishment of the HS model, cell survival rate exhibited a significant concentration-dependent relationship between Cu@CSG@CDS and CSG@CDS. When the concentration reached 150 μg / mL, the cell survival rates were 77.99% ± 1.86% and 71.17% ± 1.77%, respectively. Compared to the HS group, the cell survival rates of the Cu@CSG@CDS and CSG@CDS groups increased by approximately 30% and 20%, respectively, indicating that Cu@CSG@CDS and CSG@CDS have a therapeutic effect on NaCl-induced HCE-T cells. Furthermore, based on… Figure 12 As shown, Cu@CSG@CDS has a slight advantage in treatment efficacy compared to CSG@CDS.

[0042] 2.6 Cell viability staining analysis To further verify the protective effects of Cu@CSG@CDS and CSG@CDS on HCE-T cells subjected to oxidative stress, this invention subsequently employed fluorescent live / dead cell staining technology to further evaluate the survival status of HCE-T cells under different treatment conditions. Calcein-AM is a positively fluorescent dye capable of penetrating the membranes of living cells. In normal cells, after being hydrolyzed by esterases, it reacts with Ca... 2+ After binding, green fluorescence was detected at excitation and emission wavelengths (Ex / Em) of 514 nm / 529 nm. When intracellular esterases are absent, calcein cannot be hydrolyzed, thus it can label normal living cells. Conversely, propidium iodide (PI) cannot penetrate the membrane of normal living cells, but it easily crosses the cell membrane and binds to DNA after cell death, producing red fluorescence. Based on these properties, the combined calcein / propidium iodide (Calcein-AM / PI) staining method is often used to distinguish between living and dead cells under fluorescence microscopy. Figure 13As shown, the HS group exhibited abundant red fluorescence, revealing that HCE-T cells suffered significant death due to ROS-induced oxidative stress. In contrast, the red fluorescence intensity of the 150 μg / mL Cu@CSG@CDS combined with HS and the 150 μg / mL CSG@CDS combined with HS groups was significantly reduced compared to the HS-only treatment group, and the Cu@CSG@CDS combined with HS group was more effective than the CSG@CDS combined with HS group. This indicates a significant reduction in cell death, thus confirming that Cu@CSG@CDS and CSG@CDS are effective, and that Cu@CSG@CDS treatment is superior to CSG@CDS.

[0043] 2.7 Intracellular ROS clearance Dihydroethidium (DHE) is a commonly used fluorescent probe for superoxide anion detection, effectively detecting reactive oxygen species (ROS). DHE probes can freely enter cells and dehydrogenate under the influence of intracellular superoxide anions to form ethidium bromide. Ethidium bromide can bind to RNA or DNA, producing red fluorescence at excitation and emission wavelengths (Ex / Em) of 518 / 610 nm. Higher intracellular superoxide anion levels result in greater ethidium bromide production and stronger red fluorescence, while lower levels result in weaker fluorescence. Therefore, the properties of dihydroethidium can be used to detect superoxide anion levels. Superoxide anion levels are a type of ROS level; therefore, stronger red fluorescence intensity indicates higher ROS levels. An oxidative stress cell model was established, and DHE was used as the ROS probe. The intensity of fluorescence signals in different experimental groups was observed using a fluorescence inverted microscope. Figure 14 As shown in Figure A, HCE-T cells in the HS group exhibited strong red fluorescence and significantly increased ROS levels. After treatment with 150 μg / mL Cu@CSG@CDS and CSG@CDS, the fluorescence signals weakened to varying degrees, indicating that Cu@CSG@CDS and CSG@CDS can clear intracellular ROS. Furthermore, the fluorescence signal of the Cu@CSG@CDS nanozyme decreased more significantly, indicating that the intracellular ROS clearance effect was enhanced at this time. Figure 14 In Figure B, the ROS levels in HCE-T cells of each drug-treated group were analyzed in a semi-quantitative manner, and the results can be correlated with the fluorescence inverted image.

[0044] 2.8 Inhibitory effects of Cu@CSG@CDS and CSG@CDS on HS-induced mitochondrial dysfunction in HCE-T cells Mitochondria are the main site of ROS production in cells. JC-1, as a fluorescent indicator, selectively enters mitochondria and its fluorescence properties change with alterations in mitochondrial transmembrane potential. It was designed to study mitochondrial integrity in the context of apoptosis and monitor the dynamic changes in mitochondrial membrane potential. In healthy cells, the mitochondrial transmembrane potential is high, at which point JC-1 forms aggregates, exhibiting red / orange fluorescence. However, when mitochondria are damaged, their membrane potential decreases, and JC-1 remains in a monomeric state, exhibiting green fluorescence. This invention uses JC-1 to detect mitochondrial membrane potential damage, such as... Figure 15 As shown in Figure A, compared with the Control group, the HS group showed a significant increase in green fluorescence and almost no red fluorescence, indicating a decrease in mitochondrial membrane potential and severe mitochondrial damage. Both the 150 μg / mL HS+Cu@CSG@CDS and 150 μg / mL HS+CSG@CDS groups showed red fluorescence. Furthermore, the 150 μg / mL HS+Cu@CSG@CDS group showed weaker green fluorescence and stronger red fluorescence compared to the 150 μg / mL HS+CSG@CDS group. This indicates that Cu@CSG@CDS and CSG@CDS can effectively inhibit mitochondrial damage, and the therapeutic effect of Cu@CSG@CDS is superior to that of CSG@CDS. Figure 15 In Figure B, the level of JC-1 in HCE-T cells of each drug-treated group was analyzed in a semi-quantitative manner, and the results can be correlated with the fluorescence inverted image.

[0045] In summary, this invention successfully verified the anti-inflammatory levels of Cu@CSG@CDS and CSG@CDS at the cellular level. Through CCK8 cytotoxicity assays on HCE-T cells, hyperosmolar modeling with sodium chloride solution, post-hyperosmolar modeling treatment, uptake, Calcein-AM / PI ratio, ROS reduction, JC-1 reagent detection, and related quantitative fluorescence detection, it was shown that Cu@CSG@CDS and CSG@CDS possess good anti-inflammatory levels. Furthermore, various experiments demonstrated that at the same concentration, Cu@CSG@CDS showed superior therapeutic effects compared to CSG@CDS. The above cellular experiments also demonstrated that Cu@CSG@CDS and CSG@CDS have good biocompatibility, can be effectively taken up by cells, clear intracellular ROS, further avoid damage to mitochondrial membrane potential, and inhibit cell death.

[0046] Example 3 1. Experimental Methods 1.1 Evaluation of the in vivo pharmacodynamics of Cu@CSG@CDS and CSG@CDS 1.1.1 Establishment of the DED mouse model Thirty healthy male SPF-grade C57BL / 6J mice, weighing 20.0–22.0 g, were cultured and allowed free access to food and water before the experiment. They were housed in a 12-hour light-12-hour dark cycle for approximately one week to acclimatize. Six healthy C57 mice were randomly selected and treated with PBS buffer (pH 7.3) eye drops. The remaining mice were treated with 0.2% benzalkonium bromide (BAC) solution eye drops to induce a dry eye model. The frequency of administration was 5 μL per eye twice daily. After 7 days of induction, the success of the dry eye model was determined by measuring corneal fluorescein staining score, tear film breakup time (TBUT), and tear secretion (Schirmer test, phenol red cotton thread length) using a slit lamp.

[0047] 1.1.2 In vivo efficacy trials of Cu@CSG@CDS and CSG@CDS Sodium hyaluronate (SH), a commercially available eye drop solution, was selected as the positive control. Six healthy C57BL / 6J mice that had received eye drops in PBS buffer (pH 7.3) were divided into the Normal group. The remaining 25 healthy SPF-grade male C57BL / 6J mice with successfully established DED models were randomly divided into four groups (n=6 per group): BAC model group, BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group. The drugs were administered via intravenous instillation at a frequency of 5 μL per eye twice daily for 14 days, with post-treatment 24 hours after the last administration. Day 0 was designated as the first day of administration. On days 0, 7, 10, and 14, mice in each group received intravenous instillation of Cu@CSG@CDS, CSG@CDS, and SH, respectively, at a dose of 500 μg / mL. The Normal group and PBS model group received the same volume of physiological saline daily. On days 0, 7, 10, and 14, mouse body weight, corneal fluorescein staining score, TBUT, and Schirmer test results were observed and recorded. On day 14, mice were anesthetized via intraperitoneal injection of 2.5% tribromoethanol. After anesthesia, a circular sample was taken 1 cm from the corner of the eye, and the entire eyeball along with the corner skin was completely removed. The sample from the left eye was immediately fixed in FAS eye fixative solution. The right eye sample was rapidly transferred to an ultra-low temperature cryopreservation device at -80°C. Subsequently, the right eye sample was embedded in OCT and cryosectioned to detect changes in ROS levels in the corneal epithelial tissue.

[0048] 1.2 Evaluation of the retention effect of Cu@CSG@CDS and CSG@CDS on the ocular surface FITC was used as the fluorescent probe in this experiment. Ten healthy male C57 mice were randomly divided into two groups. Group A underwent a 7-day DED model prior to the experiment, while Group B was simultaneously instilled with PBS buffer at approximately pH 7.3. FITC-Cu@CSG@CDS solutions were prepared: Group A consisted of DED + 150 μg / mL FITC-Cu@CSG@CDS, and Group B consisted of PBS + 150 μg / mL FITC-Cu@CSG@CDS. The fluorescence intensity was measured within 40 minutes using the fluorescently labeled drug (excitation wavelength 488 nm, emission wavelength 520 nm) to assess the fluorescence intensity. In vivo optical imaging system (IVIS) experiments were performed on the mice. Eye samples were harvested, embedded using OCT, and frozen sections were prepared to observe drug retention on the ocular surface. The fluorescence intensity was quantitatively assessed using ImageJ image analysis software.

[0049] 1.3 Ocular histopathological examination The methods are as follows: ① Tissue treatment: The tissue was sequentially treated with 25% ethanol, 50% ethanol, 65% ethanol, 80% ethanol, 95% ethanol, anhydrous ethanol, and fresh anhydrous ethanol for 2 hours each. The tissue was then sequentially treated with 50% xylene and xylene. ② Paraffin infiltration and embedding: The cleared tissue was sequentially immersed in paraffin for 2, 4, and 12 hours. The paraffin-infiltrated tissue was placed in an embedding cassette, filled with paraffin, and allowed to cool until solidified to form a paraffin block. ③ Sectioning: The paraffin block was cut into 2-5 μm thick slices using a microtome, then spread in warm water at 35℃, and the spread slices were attached to glass slides and dried. ④ Dewaxing and rehydration: The tissue was dewaxed and rehydrated sequentially with xylene, anhydrous ethanol, 90% ethanol, 75% ethanol, and PBS. ⑤ Nuclear staining: The sections were stained with hematoxylin and rinsed with tap water. ⑥ Dehydration: The sections were immersed in 70% ethanol. ⑦ Cytoplasmic staining: The sections were stained with eosin. ⑧ Differentiation: Treat the stained sections with differentiation solution to remove excess dye and enhance the staining effect. ⑨ Dehydration and Clearing: Dehydrate with anhydrous ethanol and then clear with xylene. ⑩ Mounting: Mount the cleared sections with neutral resin. ⑪ Observation: Observe the tissue for pathological changes using a fluorescence upright microscope.

[0050] 1.4 Immunofluorescence assay of ocular tissue The methods are as follows: ① Pretreatment: Place the paraffin-embedded sections in a 60℃ incubator for 5 hours. ② Dewaxing and hydrating the paraffin sections: First, place the sections in xylene, then immerse them in different gradients of ethanol and deionized water three times. ③ Antigen retrieval: Add sodium citrate antigen retrieval solution, place the sections in a microwave oven, and retrieve twice on medium heat. After retrieval, allow them to cool naturally in the retrieval solution. Then rinse with deionized water. ④ Blocking endogenous peroxidase: Moisten and wash the slides with deionized water. Gently shake to remove the liquid from the surface of the slides, then use absorbent paper to remove any residual liquid around the tissue sections. Next, use a hydrophobic immunohistochemical pen to circle the tissue. ⑤ Permeabilization: Add 0.2% Trion X 100 to the sample surface, covering the entire tissue. Place the slides at room temperature for 10 minutes. After permeabilization, rinse the slides with deionized water. ⑥ Blocking: Block the tissue with 5% BSA blocking buffer at room temperature for 30 minutes, then gently shake off the liquid on the slides. ⑦ Incubation with primary antibody: Add the primary antibody to the tissue, place in a humidified chamber, and incubate overnight at 4 °C. Rinse three times with TBS. ⑧ Incubation with fluorescent secondary antibody: Add the anti-rabbit / mouse HRP-labeled polymer to the tissue, incubate at room temperature for 30-60 min, and then rinse three times with TBS. Finally, mount with anti-fluorescence quenching mounting medium and observe under a fluorescence upright microscope.

[0051] 2. Experimental Results 2.1 Corneal fluorescein score Mice in the Normal group, BAC model group, BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group were anesthetized after 0, 7, 10, and 14 days of drug administration. 2 μL of 1% sodium fluorescein solution was instilled into each eye for 1 min. The ocular surface was rinsed with PBS buffer, and the eyes were placed under a cobalt blue lamp for imaging under a slit microscope. The integrity of the corneal epithelium was evaluated according to the sodium fluorescein scoring system (commonly using a 16-point scale): the cornea was divided into 4 quadrants, each scored from 0 to 4 points. Scoring was based on the area stained with sodium fluorescein: 0 points: no staining; 1 point: sodium fluorescein staining area <5%; 2 points: sodium fluorescein staining area 5%-25%; 3 points: sodium fluorescein staining area 25%-50%; 4 points: sodium fluorescein staining area >50%. Images of the eyeballs taken under the slit microscope were compiled. The corneal epithelial tissue damage and scoring results of mice at days 0, 7, 10, and 14 are shown in the table below. Figures 16-17 .

[0052] 2.2 Analysis of Tear Film Breakup Time (TBUT) Results TBUT, or tear film breakup time, refers to the time from when the eyes are open until the first dry spot appears on the tear film. Measuring tear film breakup time is the only direct method for measuring tear film stability. Mice in the Normal group, BAC model group, BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group, after 0, 7, 10, and 14 days of drug administration, were anesthetized, stained with 1% sodium fluorescein solution, placed under cobalt blue light, and photographed under a slit microscope. The time to the appearance of the first dry spot on the tear film was observed and recorded. Figure 18 As shown, the TBUT of the Normal group was 7.5±1.0s; the TBUT of the BAC model group was 4.5±0.5s; and the TBUT of the BAC+Cu@CSG@CDS group was 5.5±1.3s, with the recovery degree being closest to that of the Normal group. Figure 19 The graph shows the differences in mouse body weight at 0, 7, 10, and 14 days after drug administration. Except for the Normal group, which showed a significant increase in body weight, the body weight of mice in the other groups remained relatively stable, demonstrating that the drug did not affect the normal living conditions of the mice.

[0053] 2.3 Analysis of the results of the tear secretion test (Schirmer test) Phenolic red cotton thread was pre-cut into segments of uniform length. Mice in the Normal group, BAC model group, BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group were anesthetized after 0, 7, 10, and 14 days of drug administration. The test strip was gently placed in the middle and outer third of the lower eyelid, left for 15 seconds, and then the phenol red cotton thread was removed. The length of the discolored area was measured and recorded using a ruler. By measuring the tear fluid data on day 14 in the Normal group, BAC model group, BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group, it was observed that the tear secretion in the PBS group was the worst. The tear secretion in the BAC+Cu@CSG@CDS group, BAC+CSG@CDS group, and BAC+SH group improved. On day 14, the recovery level of the BAC+Cu@CSG@CDS group and the BAC+CSG@CDS group was close to that of the Normal group. Figure 20 This is a statistical graph showing the tear secretion of mice on day 14 in the Schirmer test.

[0054] 2.4 Analysis of ROS Levels in Corneal Epithelial Tissue Eyeball samples were embedded in OCT, frozen sections were prepared, and corneal epithelial tissue was stained with DHE and photographed under an upright fluorescence microscope. Figure 21From left to right: Normal group, BAC model group, BAC+SH group, BAC+CSG@CDS group, BAC+Cu@CSG@CDS group. The red fluorescence intensity gradually decreases, indicating that the BAC+Cu@CSG@CDS group has the best treatment effect, followed by the BAC+SH group and the BAC+CSG@CDS group. Figure 22 To semi-quantitatively analyze the ROS levels in corneal epithelial tissue of each drug-treated group, the results can be correlated with positive fluorescence images.

[0055] 2.5 Analysis of mouse in vivo imaging results To observe drug retention in the corneal epithelial tissue of normal healthy mice and mice with dry eye syndrome, 20 healthy male C57BL / 6J mice were selected. Ten mice were randomly chosen and treated with PBS buffer at approximately pH 7.3 as the Normal group. The remaining mice were treated with 0.2% BAC solution to induce a dry eye model as the DED group. FITC-Cu@CSG@CDS was prepared. After anesthesia, in vivo fluorescence imaging was performed, and eyeballs were harvested at time points. Drug retention on the ocular surface was assessed based on the in vivo imaging results. Figure 23 As shown, FITC-Cu@CSG@CDS can be detected with strong fluorescence on the ocular surface. Within 40 min, the fluorescence intensity of both the Normal group and the DED group showed a decreasing trend with increasing time. In comparison, within 10 min to 40 min, the fluorescence intensity of the ocular surface of mice in the DED group was higher than that in the Normal group, indicating that the amount of FITC-Cu@CSG@CDS retained on the ocular surface of the DED group was higher than that in the Normal group. Figure 24 Quantitative statistical analysis of fluorescence ROI in live imaging images. Figure 25 The retention status of FITC-Cu-CSG-CDs at different time periods conforms to this trend.

[0056] 2.6 Analysis of histopathological staining results of corneal epithelial tissue To observe the recovery of corneal epithelial tissue in mice with dry eye after drug treatment, eyeball samples were paraffin-embedded and stained with hematoxylin and eosin (HE). The condition of the corneal tissue was observed and recorded, and pathological sections were photographed under an upright microscope. Figure 26As shown, with the corneal epithelial tissue state of the Normal group as a control, the corneal surface of normal healthy mice has corneal epithelial cells arranged in a tight and orderly manner. Mice after modeling have obvious characteristics of corneal epithelial defects and reduced number of goblet cells. The arrangement and morphology of the corneal epithelium in the BAC model group are disrupted, and the corneal stroma is loose and thickened. The BAC+SH group and the BAC+CSG@CDS group have a mild recovery state, and the corneal epithelial cell layer is rearranged in a regular and orderly manner. The BAC+Cu@CSG@CDS group has significantly restored the morphology and structure of the corneal epithelium, and the corneal epithelial layer is tightly arranged, close to the state of the Normal group. HE sections of the retina show that CSG@CDS, Cu@CSG@CDS, and SH have no retinal toxicity and are safe for ocular use. To further investigate the in vivo biosafety of CSG@CDS, Cu@CSG@CDS, and SH, H&E staining was performed on sections of heart, liver, spleen, lung, and kidney tissues from mice. All organ tissues showed intact structures and no abnormalities such as inflammation, necrosis, congestion, or hemorrhage were observed. Figure 27 This indicates that CSG@CDS, Cu@CSG@CDS, and SH have good tissue safety and no toxic side effects.

[0057] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme, characterized in that, Includes the following steps: Copper ions, *Sargentodoxa cuneata*, and water were mixed and subjected to a hydrothermal reaction. The supernatant was then obtained by centrifugation. After filtration, dialysis and drying of the supernatant, the copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme was obtained.

2. The preparation method according to claim 1, characterized in that, The hydrothermal reaction was carried out at a temperature of 180°C.

3. The preparation method according to claim 1, characterized in that, The hydrothermal reaction time is 8 hours.

4. The preparation method according to claim 1, characterized in that, The mass ratio of copper ions to *Sargentodoxa cuneata* is 1:

10.

5. The preparation method according to claim 1, characterized in that, The filtration process uses a 0.22 μm filter membrane.

6. The preparation method according to claim 1, characterized in that, The dialysis process uses a 400 Da dialysis bag.

7. A copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme prepared by the preparation method according to any one of claims 1-6.

8. The application of the copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme as described in claim 7 in the preparation of a drug for treating dry eye syndrome.

9. A drug for treating dry eye syndrome, characterized in that, The active ingredient includes the copper ion-Sargentodoxa cuneata carbon quantum dot nanozyme as described in claim 7.

10. The medicament according to claim 9, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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