Nano-enzyme M-SA / NC-coated CDs, eye drops and preparation method and application of nano-enzyme M-SA / NC-coated CDs

Abstract

The invention belongs to the technical field of fungal keratitis, and discloses a nano-enzyme M-SA / NC-coated CDs, eye drops, and a preparation method and application of the nano-enzyme M-SA / NC-coated CDs. The nano enzyme M-SA / NC coated CDs is positive charge carbon dots co-modified by M single atoms and M nano clusters, wherein M represents an antibacterial metal element. The preparation method comprises the following steps: (1) carrying out ultrasonic treatment on L-arginine, a water-soluble M salt, water and a cationic quaternary ammonium salt aqueous solution until a clear solution is obtained; (2) heating the obtained solution to the reaction temperature of 180-240 DEG C, keeping the temperature for 2-6 hours, and obtaining a viscous product after the reaction is finished; (3) ultrasonically dispersing the obtained viscous product in water, then centrifuging, and putting supernate in a dialysis bag; and immersing the dialysis bag in water for dialysis, and performing vacuum freeze drying after dialysis to obtain M-SA / NC-coated CDs powder. The invention also discloses an application of the nano enzyme M-SA / NC coated CDs in preparation of a medicine for treating fungal keratitis. The nano enzyme M-SA / NC coated CDs disclosed by the invention can maintain the in-vivo anti-inflammatory property for a long time while exerting an antifungal effect, and has a relatively good treatment effect in vivo and in vitro.

Description

Technical Field

[0001] This invention belongs to the field of fungal keratitis technology, specifically relating to a nanozyme M-SA / NC@CDs, eye drops, its preparation method, and its application. Background Technology

[0002] Fungal keratitis (FK) is a serious infectious ocular surface disease that threatens vision. It can lead to corneal perforation and even endophthalmitis, often resulting in enucleation if the infection cannot be controlled. Even if the infection is controlled, it often leads to scarring and clouding of the corneal tissue, severely affecting vision. Global FK incidence statistics in 2021 showed 1-1.4 million new cases annually, with 8-11% of patients requiring enucleation. FK is a pathological process where fungal infection causes corneal tissue damage, inducing extensive inflammatory cell infiltration, followed by corneal tissue healing. The only approved clinically used drug, natamycin eye drops, is a suspension with very poor corneal permeability and is essentially ineffective against deep corneal infections. Currently, FK is facing a situation where no effective treatment is available.

[0003] The root cause of this situation is the presence of local drug delivery barriers in the eye (tear film barrier and corneal barrier), which hinder drugs from reaching the site of fungal infection. These two barriers reduce the bioavailability of many drugs in the eye to only 1-7%. Frequent use of clinical drugs increases toxic side effects and easily leads to drug resistance, significantly reducing the therapeutic effect of antibiotics. Therefore, after FK infection, the combined influence of the above factors makes the infection difficult to control. Even if the infection is controlled, excessive inflammation causing corneal stromal fibrosis still leads to vision loss. Therefore, how to overcome the ocular drug delivery barrier to rapidly and efficiently kill fungi while inhibiting inflammation and effectively preventing corneal blindness is a pressing problem that needs to be explored and solved. Exploring new materials or novel drug delivery systems that can overcome the drug delivery barrier is key to solving this problem. Summary of the Invention

[0004] To address the three major challenges faced by FK drug therapy in clinical practice—poor corneal permeability, inadequate antifungal efficacy, and inability to simultaneously reduce inflammation associated with infection—this invention aims to provide a nanozyme M-SA / NC@CDs, eye drops, its preparation method, and its application.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A nanozyme M-SA / NC@CDs, wherein the nanozyme M-SA / NC@CDs is a positively charged carbon dot co-modified with M single atoms and M nanoclusters, wherein M represents an antibacterial metal element.

[0006] Preferably, M is Cu, Ag, Ce, Fe, Mn, or Co.

[0007] A method for preparing nanozymes M-SA / NC@CDs, the preparation steps are as follows: (1) Sonicate L-arginine, water-soluble M salt, water and cationic quaternary ammonium salt aqueous solution together until a clear solution is obtained; wherein, the concentration of the cationic quaternary ammonium salt aqueous solution is 50~90wt%; the water-soluble M salt is calculated based on the M ion therein, and the raw material ratio is L-arginine: water-soluble M salt: water: cationic quaternary ammonium salt aqueous solution = 0.6g: (0.4139~0.8278)mmol: (4~8)mL: (1~2)mL; (2) Heat the solution obtained in step (1) to the reaction temperature of 180~240℃ and maintain this temperature for 2~6h. After the reaction is completed, a viscous product is obtained. (3) The viscous product obtained in step (2) is ultrasonically dispersed in water and then centrifuged. The supernatant obtained by centrifugation is placed in a dialysis bag. The dialysis bag is immersed in water for dialysis. After dialysis, it is freeze-dried under vacuum to obtain M-SA / NC@CDs powder.

[0008] Preferably, in step (1), the water-soluble M salt is a nitrate.

[0009] Preferably, in step (1), the cationic quaternary ammonium salt is dimethyl diallyl ammonium chloride.

[0010] Preferably, in step (2), the temperature is heated to the reaction temperature at a rate of 4~10℃ / min.

[0011] Preferably, in step (3), the centrifugation speed is 10000~15000 rpm and the time is 10~30 min.

[0012] Preferably, in step (3), the molecular weight cutoff of the dialysis bag is 500~3000 Da and the total dialysis time is 24~72 h.

[0013] Application of a nanozyme M-SA / NC@CDs in the preparation of drugs for treating fungal keratitis.

[0014] An antifungal eye drop comprising the nanozyme M-SA / NC@CDs, the antifungal eye drop comprising nanozyme M-SA / NC@CDs and water for injection, wherein the mass-to-volume ratio of nanozyme M-SA / NC@CDs to water for injection is at least 7.8 μg / mL.

[0015] Beneficial effects: This invention prepared nanozymes M-SA / NC@CDs, which were applied to the preparation of antifungal eye drops. The prepared eye drops are easy to use and can kill Fusarium solani, the main fungal species causing fungal keratitis. At the same time, it reduces the expression of inflammatory factors (NF-κB, P-NF-κB p65, TNF-α, IL-1β and IL-6) and promotes the upregulation of the expression of anti-inflammatory factor IL-10. While exerting antifungal effects, it also maintains long-term anti-inflammatory properties in vivo, showing good therapeutic effects both in vivo and in vitro. Attached Figure Description

[0016] Figure 1 Characterization results of Cu-SA / NC@CDs prepared for Example 1: (A) Transmission electron microscopy (TEM) images of Cu-SA / NC@CDs; (B) High-resolution transmission electron microscopy (HR-TEM) images; (C) Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (ac-HAADF-STEM) images of Cu-SA / NC@CDs; (DF) XPS fine spectra of C, O, and N elements in Cu-SA / NC@CDs; (G) K-edge X-ray absorption near-edge structure spectra of Cu-SA / NC@CDs and reference samples Cu foil and CuO; (H) Fourier transform extended X-ray absorption fine structure spectra of Cu-SA / NC@CDs and reference samples Cu foil and CuO (FT-EXAFS); (I) Hydrodynamic diameter distribution and Zeta potential of Cu-SA / NC@CDs (inset).

[0017] Figure 2 For in vitro antifungal effects: (A) Inhibitory efficiency of different concentrations of Cu-SA / NC@CDs against Fusarium solani; (B) Fungal activity of Cu-SA / NC@CDs against Fusarium solani at different time points; (C) Colony images of Fusarium solani after treatment with Cu-SA / NC@CDs at 5 times the minimum inhibitory concentration (5×MIC) for different time periods; (D) Scanning electron microscopy (SEM) images of hyphae and spores in different treatment groups; (E) Fluorescent images of live / dead fungi stained in different treatment groups.

[0018] Figure 3 Immunofluorescence staining images (A) and quantitative analysis (B) of CAT, SOD1, GPX1, TNF-α, IL-6, IL-1β, IL-10, NF-κB and P-NF-κB p65.

[0019] Figure 4Results of in vivo antifungal treatment: (A) Representative corneal photographs of mice with keratitis treated in different treatment groups; (B) Slit-lamp photomicrographs of mice with keratitis treated in different treatment groups; (C) Images of mice with keratitis treated in different treatment groups after fluorescein sodium staining; (D) Representative corneal tissue sections (hexaammonium silver staining) of mice in different treatment groups 3 days after treatment; (E) Hematoxylin-eosin (HE) staining histological examination of corneal tissue sections of mice in different treatment groups and at different time points (day 3 and day 7); (F) Clinical scores of mice with keratitis in different treatment groups; (G) and (H) Corneal thickness measured in different treatment groups on day 3 and day 7, respectively. Detailed Implementation

[0020] To enable those skilled in the art to better understand the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0021] Example 1

[0022] A method for preparing Cu-SA / NC@CD nanozymes, the preparation steps are as follows: (1) Sonicate 0.6g L-arginine, 0.2g CuNO3·3H2O, 4mL deionized water and 1mL dimethyl diallyl ammonium chloride aqueous solution (50wt%) together for 5min to obtain a clear solution; (2) Heat the solution obtained in step (1) to the reaction temperature of 240°C at a rate of 4°C / min and maintain this temperature for 3 hours. After the reaction is completed, a viscous product is obtained. (3) Add the viscous product obtained in step (2) to 20 mL of deionized water, sonicate for 30 min, and then centrifuge at 15000 rpm for 30 min. Place the supernatant obtained by centrifugation into a dialysis bag with a molecular weight cutoff of 500 Da. Immerse the dialysis bag in water for dialysis for 24 h. After dialysis, freeze dry under vacuum to obtain Cu-SA / NC@CDs powder.

[0023] Product structure characterization Figure 1Characterization results of Cu-SA / NC@CDs prepared for Example 1: (A) Transmission electron microscopy (TEM) images of Cu-SA / NC@CDs; (B) High-resolution transmission electron microscopy (HR-TEM) images; (C) Aberration-corrected high-angle annular dark-field scanning transmission electron microscopy (ac-HAADF-STEM) images of Cu-SA / NC@CDs; (DF) XPS fine spectra of C, O, and N elements in Cu-SA / NC@CDs; (G) K-edge X-ray absorption near-edge structure spectra of Cu-SA / NC@CDs and reference samples Cu foil and CuO; (H) Fourier transform extended X-ray absorption fine structure spectra of Cu-SA / NC@CDs and reference samples Cu foil and CuO (FT-EXAFS); (I) Hydrodynamic diameter distribution and Zeta potential of Cu-SA / NC@CDs (inset).

[0024] Figure 1 A shows that Cu-SA / NC@CDs are well-dispersed near-spherical nanoparticles with an average diameter of approximately 3.02 nm.

[0025] Figure 1 B shows that there are lattice stripes with a spacing of 0.20325 nm, corresponding to the (101) crystal plane, which proves that there is a graphite-like structure in Cu-SA / NC@CDs.

[0026] Figure 1 C shows that Cu-SA / NC@CDs are uniformly distributed with an average planar size of 2 nm; ac-HAADF-STEM images confirm that the isolated copper atoms are anchored on a nitrogen-doped carbon-based support. Figure 1 The dispersion of small bright spots in Cu-SA / NC@CDs was characterized, suggesting they may be individual copper atoms or copper nanoclusters.

[0027] Figure 1 The D-display shows that the secondary peaks at 288.1 eV and 285.8 eV in the C 1s region indicate that Cu-SA / NC@CDs contain hydroxyl (COC), carbonyl (C=O) and C=N bonds.

[0028] Figure 1 E shows that the O 1s spectrum is decomposed into three peaks at 531.6 eV and 533.2 eV, which are attributed to C=O and COH, respectively. The presence of O can be attributed to adsorbed oxygen-containing functional groups.

[0029] Figure 1F shows that the N 1s spectrum decomposes into four peaks: the peak at 401.5 eV is graphite nitrogen (graphite-N), the peak at 400.5 eV is pyrrolidine nitrogen (pyrrolidine-N), the peak at 399.6 eV is pyridine nitrogen (pyridine-N), and the peak at 398.4 eV is Cu-N.

[0030] Figure 1 G shows that the absorption edge position of Cu in Cu-SA / NC@CDs (~8977eV) is between Cu foil and CuO, indicating that the valence state of Cu in Cu-SA / NC@CDs is between 0 and +2.

[0031] Figure 1 The H-value shows that the main peak at 1.48 Å belongs to the Cu-N scattering path, corresponding to the first coordination shell of Cu-N; the peak at 2.45 Å belongs to the Cu-Cu scattering path.

[0032] The hydration particle size and potential of Cu-SA / NC@CDs were determined using a Malvern laser particle size analyzer. Figure 1 I shows that the hydrated particle size of Cu-SA / NC@CDs is 4.53 nm, which is basically consistent with the transmission electron microscopy image; the zeta potential shows that the surface charge of Cu-SA / NC@CDs is +10.5 mV ( Figure 1 I. Illustration).

[0033] The above results confirm that the synthesis of Cu-SA / NC@CDs was successful.

[0034] Performance testing I. In vitro antifungal performance test Fusarium solani, a common cause of clinical ocular infections, was selected as the research subject. This strain was isolated from patients with fungal keratitis (the isolation technique can refer to existing techniques, such as: Yang Huiying et al. Diagnosis and identification of pathogenic filamentous fungal infections of the eye [J]. Chinese Journal of Nosocomial Infection, 2002, Vol. 12, No. 5).

[0035] (1.1) Inhibition efficiency of different concentrations of Cu-SA / NC@CDs against Fusarium solani After the isolated Fusarium solani fungal spores were cultured on Sabouraud dextrose agar at 28°C for 48 h, the spores were inoculated into 96-well plates containing 100 μL of RPMI 1640 medium, with 10 spores per well. 4Spores were collected, and then different volumes of Cu-SA / NC@CDs aqueous solution (1 mg / mL) were added to different wells of a 96-well plate, so that the final concentrations of Cu-SA / NC@CDs in the wells were 0, 1, 2, 4, 7.8, 15.6, 31.52, 62.5, 125 and 250 μg / mL, respectively. After incubation at 28°C for 48 h, the OD600 values ​​were recorded using a Cytation5 microplate reader (Biotek Winooski, USA).

[0036] The results are as follows Figure 2 As shown in Figure A, fungal growth was inhibited to varying degrees with increasing Cu-SA / NC@CDs concentration. When the Cu-SA / NC@CDs concentration was 7.8 μg / mL (minimum inhibitory concentration, MIC), more than 90% of fungal growth was inhibited.

[0037] (1.2) Evaluation of the fungal activity of Cu-SA / NC@CDs against Fusarium solani at different time points After the isolated Fusarium solani fungal spores were cultured on Sabouraud agar at 28°C for 48 hours, the fungal spores were then introduced at an initial concentration of 10... 4The cells / mL were inoculated into RPMI 1640 medium and cultured for 8 hours under a shaker at 170 rpm. Then, they were divided into four groups: (1) Control group: an equal volume of RPMI 1640 medium was added to the culture medium; (2) 5×MIC group: an equal volume of Cu-SA / NC@CDs aqueous solution with a concentration of 78 μg / mL was added to the culture medium (by diluting Cu-SA / NC@CDs aqueous solution with a concentration of 1 mg / mL using RPMI 1640 medium) so that the final concentration of Cu-SA / NC@CDs reached 5MIC, i.e., 39 μg / mL; (3) 2×MIC group: an equal volume of Cu-SA / NC@CDs aqueous solution with a concentration of 31.2 μg / mL was added to the culture medium (by diluting Cu-SA / NC@CDs aqueous solution with a concentration of 1 mg / mL using RPMI 1640 medium) so that the final concentration of Cu-SA / NC@CDs reached 5MIC, i.e., 39 μg / mL; (4) 2×MIC group: an equal volume of Cu-SA / NC@CDs aqueous solution with a concentration of 31.2 μg / mL was added to the culture medium (by diluting Cu-SA / NC@CDs aqueous solution with a concentration of 1 mg / mL using RPMI 1640 medium) so that the final concentration of Cu-SA / NC@CDs reached 5MIC, i.e., 39 μg / mL. (4) Vor group: Add an equal volume of voriconazole aqueous solution with a concentration of 117.6 μg / mL to the culture medium (by diluting voriconazole eye drops with a concentration of 1 mg / mL using RPMI 1640 medium) to the culture medium to make the final concentration of voriconazole 58.8 μg / mL. Subsequently, samples were taken from each of the above groups at different time intervals (0, 0.5, 1, 2, 4, 6 and 8 h), and the fungal solution was diluted 100 times by the dilution plating method. 100 μL of the diluted solution was spread on Sabouraud dextrose agar (SDA) plates for colony counting. The value was the average of at least three biological replicates (in at least three independent experiments).

[0038] The results of fungal activity of Cu-SA / NC@CDs against Fusarium solani at different time points are as follows: Figure 2 As shown in Figure B, the number of fungal colonies gradually decreased with increasing drug concentration and culture time. Furthermore, at the same time point, the bactericidal effects of Cu-SA / NC@CDs in the 2×MIC and 5×MIC groups were significantly better than those in the voriconazole group. Colony images of *F. solani* after treatment with Cu-SA / NC@CDs at 5 times the minimum inhibitory concentration (5×MIC) for different times are shown in Figure B. Figure 2 As shown in Figure C, the results indicate that when Fusarium solani was treated with Cu-SA / NC@CDs for 6 hours, no fungal colony growth was observed on Sabouraud dextrose agar plates, demonstrating the most significant antifungal effect.

[0039] (1.3) Damage to Fusarium solani spores and cell membranes by Cu-SA / NC@CDs The isolated Fusarium solani fungal spores were cultured on Sabouraud agar at 28°C for 48 hours. The spores were then introduced at an initial concentration of 10...4 Inoculated at 1 / mL concentration into RPMI 1640 medium and cultured for 8 h at 170 rpm. Then, the cells were divided into three groups: Control group: an equal volume of RPMI 1640 medium was added to the culture medium; Cu-SA / NC@CDs group: an equal volume of 78 μg / mL Cu-SA / NC@CDs aqueous solution (prepared by diluting 1 mg / mL Cu-SA / NC@CDs aqueous solution with RPMI 1640 medium) was added to the culture medium, bringing the final Cu-SA / NC@CDs concentration to 39 μg / mL; Vor group: an equal volume of 117.6 μg / mL voriconazole aqueous solution (prepared by diluting 1 mg / mL voriconazole eye drops with RPMI 1640 medium) was added to the culture medium, bringing the final voriconazole concentration to 58.8 μg / mL. Each group was treated for 4 hours. After h, the sample was fixed on a glass slide with 4% glutaraldehyde, washed with PBS, and dried overnight. After gold sputtering, the damage to fungal spores and cell membranes was observed using scanning electron microscopy.

[0040] Scanning electron microscope (SEM) images of hyphae and spores in different treatment groups are shown below. Figure 2 As shown in D, the scale bar is 2 μm. The results showed that the spores and hyphae treated with Cu-SA / NC@CDs exhibited disordered cell morphology, with varying degrees of surface wrinkling, depression, perforation and damage, and severe disruption of structural integrity. In contrast, the spores and hyphae in the Control group and those treated with voriconazole maintained intact structures, and no obvious cell membrane damage was observed.

[0041] The results above show that Cu-SA / NC@CDs have excellent in vitro antifungal activity.

[0042] (1.4) Staining of live / dead bacteria The isolated Fusarium solani fungal spores were cultured on Sabouraud agar at 28°C for 48 hours. The spores were then introduced at an initial concentration of 10... 4Inoculated at 1 / mL concentration into RPMI 1640 medium and cultured for 8 h at 170 rpm. Then, the cells were divided into three groups: Control group: an equal volume of RPMI 1640 medium was added to the culture medium; Cu-SA / NC@CDs group: an equal volume of 78 μg / mL Cu-SA / NC@CDs aqueous solution (prepared by diluting 1 mg / mL Cu-SA / NC@CDs aqueous solution with RPMI 1640 medium) was added to the culture medium, bringing the final Cu-SA / NC@CDs concentration to 39 μg / mL; Vor group: an equal volume of 117.6 μg / mL voriconazole aqueous solution (prepared by diluting 1 mg / mL voriconazole eye drops with RPMI 1640 medium) was added to the culture medium, bringing the final voriconazole concentration to 58.8 μg / mL. Each group was treated for 4 hours. After h, the live / dead bacteria staining kit was operated according to the instructions of the kit (Syto9-PI method, Tianjingsha; catalog number: 11-MK220950-40) under room temperature and dark conditions. After mixing and incubating for 15 min, excess dye was removed by centrifugation. 10 μL of the stained fungal suspension was placed on a glass slide. Finally, the stained fungal images were observed using a fluorescence microscope.

[0043] Fluorescence images of live / dead fungi stained in different treatment groups, as shown below. Figure 2 As shown in Figure E, the scale bar is 20 μm. The results showed that fungal spores in the Control group fluoresced in the green channel but did not fluoresce in the red channel, indicating that the untreated fungi had intact cell membranes and remained active. In contrast, the Cu-SA / NC@CDs treated group showed a strong fluorescence signal in the red channel, which was basically consistent with the spores under the green fluorescence signal, indicating that the cell membranes of most fungi were severely damaged after Cu-SA / NC@CDs treatment, suggesting that Cu-SA / NC@CDs can damage the cell membranes of fungi.

[0044] II. Anti-inflammatory performance test HCE-2 cells were distributed at a rate of 5 × 10⁶ cells per well. 3Inoculated at a density of 1000 cells / well with coverslips placed in the wells of a six-well plate, and cultured in DMEM / F12 complete medium at 37°C and 5% CO2 for 24 h. Subsequently, the plates were divided into three groups: Control group: the old medium was discarded, and freshly prepared DMEM / F12 complete medium was added for 6 h, followed by another 24 h of treatment with the same medium. Cu-SA / NC@CDs group: the old medium was discarded, and serum-free DMEM / F12 medium containing Cu-SA / NC@CDs (2 µg / mL) was added for 6 h of pretreatment, followed by another 6 h of treatment with the same medium and then hypertonic medium (DMEM / F12 complete medium with added sodium chloride to adjust the osmotic pressure to 500 Ω·cm). The mOsM group was cultured for 24 h; the HOM group: the old medium was discarded, and freshly prepared DMEM / F12 complete medium was added for 6 h, then the old medium was discarded and hypertonic medium (as above) was added for 24 h; when discarding the old medium in each group, the amount of medium added afterward was equal to the amount of old medium discarded; after the treatment of each group, the cells were fixed with 4% paraformaldehyde (Biosharp, China) for 10 min, washed, permeabilized with 0.5% Triton X-100 for 20 min, and blocked with goat serum (Elabscience, China) at room temperature for 30 min; then, HCE-2 cells were incubated overnight at 4 ℃ with primary antibodies against CAT, SOD1, GPX1, TNF-α, IL-6, IL-1β, IL-10, NF-κB, and P-NF-κB p65; the next day, the cells were incubated with Cy3-labeled goat anti-rabbit IgG under light-protected conditions. Incubate with H&L secondary antibody at room temperature for 1 hour, counterstain the nucleus with DAPI, and acquire images using a fluorescence microscope (Nikon ECLIPSE 80i, Japan).

[0045] Figure 3 Immunofluorescence staining images (A) and quantitative analysis (B) of CAT, SOD1, GPX1, TNF-α, IL-6, IL-1β, IL-10, NF-κB and P-NF-κB p65. Scale bar: 10 μm. Data are expressed as mean ± standard deviation. *p<0.05, **p<0.01, ***p<0.001 and NS p>0.05.

[0046] The results showed that compared with the HOM group, the activities of SOD1, CAT, and GPX1 were significantly increased in the Cu-SA / NC@CDs group. Cu-SA / NC@CDs effectively counteracted the decrease in antioxidant enzyme activity caused by hyperosmolarity. Simultaneously, Cu-SA / NC@CDs significantly reduced the mean fluorescence intensity (MFI) of hyperosmolarity-induced pro-inflammatory factors NF-κB, P-NF-κB p65, TNF-α, IL-1β, and IL-6, and increased the expression of the anti-inflammatory factor IL-10 induced by hyperosmolarity. These results fully demonstrate that Cu-SA / NC@CDs can effectively eliminate inflammation caused by fungal infection.

[0047] III. Experimental Treatment of Fungal Keratitis The isolated Fusarium solani fungal spores were cultured on Sabouraud agar at 28°C for 48 hours. The spores were then introduced at an initial concentration of 10... 4 Inoculate 10⁶ cells / mL into RPMI 1640 medium and continue culturing at 170 rpm for 8 h to obtain a suspension of Fusarium solani fungal spores (10⁶ cells / mL). 4 (CFU / mL).

[0048] Twenty-four healthy C57BL / 6 mice were selected, each with a normal ocular surface, free from lesions such as cataracts, opacities, and leukoplakia. Six mice were selected as the Normal group, and 18 mice were used for modeling. The modeling steps were as follows: First, the mice were anesthetized, their whiskers were trimmed, and the skin around the eyes was disinfected with povidone-iodine. Then, the corneal surface of the mice was anesthetized with promecaine hydrochloride eye drops (15mL:75mg) to ensure complete exposure of the cornea. Second, under a surgical microscope (Topcon OMS-90, Japan), the right cornea was marked with a sterile trephine with a diameter of 2mm, while the left eye was left untreated. Then, the corneal epithelium within the marked circular area was scraped off with a sterile 45-gauge blade. Next, cross-scratches were made on the mouse cornea to a depth reaching the superficial stroma layer using the "cross-scratching method". Subsequently, 5μL of Fusarium solani fungal spore suspension (10 4 A fungal keratitis model was established by instilling 5 μL of Cu-SA / NC@CDs solution into the eyes of mice. Twelve hours after modeling, 18 mice were randomly divided into three groups: a Control group (physiological saline), a Cu-SA / NC@CDs group (25 μg / mL Cu-SA / NC@CDs aqueous solution), and a Vor group (25 μg / mL voriconazole aqueous solution, prepared by diluting 1 mg / mL voriconazole eye drops with physiological saline), with six mice in each group. All groups received the drug via eye drops, 5 μL three times daily for seven consecutive days.

[0049] Observation: Day 0 was 12 hours after modeling. Slit-lamp photography was used, and fluorescein staining was employed to observe the modeling progress in each mouse group, the uniformity of lesions between groups, and to report findings based on relevant literature. Invest Ophthalmol Vis Sci Clinical scoring was performed using the scoring criteria (2003, 44, 210). Following drug administration, corneal pathological changes were observed using a slit lamp on days 1, 3, 5, and 7, with photographs recorded and scored by experienced clinicians.

[0050] Pathological examination: During the course of fungal keratitis in mice, mouse eyeballs were collected on days 3 and 7, fixed with FAS eye fixative for 2 days, embedded in paraffin, and cut into 5 μm thick sections for pathological examination. Corneal tissues on days 3 and 7 were observed under an optical microscope using hematoxylin and eosin (H&E) staining to assess tissue lesions and repair. Hexaammonium silver staining was used to assess the growth and invasion of fungal spores and hyphae in corneal tissue. Immunofluorescence staining was used to assess the expression levels of inflammatory factors in the corneal tissue of mice after treatment.

[0051] Figure 4 Results of in vivo antifungal treatment: (A) Representative corneal photographs of mice with keratitis treated in different treatment groups; (B) Slit-lamp photomicrographs of mice with keratitis treated in different treatment groups; (C) Images of mice with keratitis treated in different treatment groups after fluorescein sodium staining; (D) Representative corneal tissue sections (hexaammonium silver staining) of mice in different treatment groups 3 days after treatment; (E) Hematoxylin-eosin (HE) staining histological examination of corneal tissue sections of mice in different treatment groups and at different time points (day 3 and day 7); (F) Clinical scores of mice with keratitis in different treatment groups; (G) and (H) Corneal thickness measured in different treatment groups on day 3 and day 7, respectively. Data are expressed as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001, NSp>0.05.

[0052] Figure 4 A, B, and C show that all three groups of mice exhibited corneal infiltration and corneal edema. In the Control group, mixed conjunctival hyperemia and corneal neovascularization occurred throughout the disease progression, with corneal perforation occurring on day 3, indicating severe corneal infection. Compared to the Control group, the treatment groups (especially the Cu-SA / NC@CDs group) effectively inhibited fungal infection of the mouse cornea throughout the treatment process. Although the Vor group showed incomplete epithelial repair and mild corneal edema, it still demonstrated a therapeutic effect. When Cu-SA / NC@CDs were used continuously for 7 days, the cornea became significantly clearer, and the corneal epithelium basically recovered its integrity.

[0053] Figure 4The results showed that a large number of fungi were found in the corneal stroma of mice in the Control group, indicating a severe fungal infection; a small number of fungi were still present in the corneal tissue of mice in the Vor group; however, no significant fungal growth was observed in the Cu-SA / NC@CDs group compared with the Control and Vor groups; these results indicate that Cu-SA / NC@CDs can completely inhibit the growth of fungi in the FK of mice.

[0054] Figure 4 E and G showed that on day 3, the Control group had extensive inflammatory cell infiltration in the corneal tissue, with significant thickening and edema. Compared with the Control group, the treatment group had fewer inflammatory cells, with only mild corneal edema. Furthermore, the Cu-SA / NC@CDs group had fewer inflammatory cells than the Vor group, and other clinical manifestations were alleviated. In addition, H&E staining images showed that the Vor group had significant corneal defects, while the corneal epithelium in the Cu-SA / NC@CDs group showed only mild damage.

[0055] Figure 4 E and H showed that on day 7, abnormal corneal structures with high inflammatory cell infiltration were observed in the corneal stroma of the Control group. This immune response could further induce endophthalmitis and corneal perforation. In contrast, mice in the Vor group treated for 7 days showed a milder inflammatory response, while the corneal tissue of mice in the Cu-SA / NC@CDs group basically returned to normal.

[0056] Figure 4 F shows that Cu-SA / NC@CDs have better efficacy and lower clinical scores in vivo, and significantly shorten the course of corneal transplant rejection (FK).

Claims

1. A nanozyme M-SA / NC@CDs, characterized in that: The nanozyme M-SA / NC@CDs is a positively charged carbon dot co-modified with M single atoms and M nanoclusters, where M represents an antibacterial metal element.

2. The nanozyme M-SA / NC@CDs as described in claim 1 or 2, characterized in that: M is Cu, Ag, Ce, Fe, Mn, or Co.

3. A method for preparing the nanozyme M-SA / NC@CDs as described in claim 1 or 2, characterized in that, The preparation steps are as follows: (1) Sonicate L-arginine, water-soluble M salt, water and cationic quaternary ammonium salt aqueous solution together until a clear solution is obtained; wherein, the concentration of the cationic quaternary ammonium salt aqueous solution is 50~90wt%; the water-soluble M salt is calculated based on the M ion therein, and the raw material ratio is L-arginine: water-soluble M salt: water: cationic quaternary ammonium salt aqueous solution = 0.6g: (0.4139~0.8278)mmol: (4~8)mL: (1~2)mL; (2) Heat the solution obtained in step (1) to the reaction temperature of 180~240℃ and maintain this temperature for 2~6h. After the reaction is completed, a viscous product is obtained. (3) The viscous product obtained in step (2) is ultrasonically dispersed in water and then centrifuged. The supernatant obtained by centrifugation is placed in a dialysis bag. The dialysis bag is immersed in water for dialysis. After dialysis, it is freeze-dried under vacuum to obtain M-SA / NC@CDs powder.

4. The method for preparing the nanozyme M-SA / NC@CDs as described in claim 3, characterized in that: In step (1), the water-soluble M salt is a nitrate.

5. The method for preparing the nanozyme M-SA / NC@CDs as described in claim 3, characterized in that: In step (1), the cationic quaternary ammonium salt is dimethyl diallyl ammonium chloride.

6. The method for preparing the nanozyme M-SA / NC@CDs as described in claim 3, characterized in that: In step (2), the temperature is heated to the reaction temperature at a rate of 4~10℃ / min.

7. The method for preparing the nanozyme M-SA / NC@CDs as described in claim 3, characterized in that: In step (3), the centrifugation speed is 10000~15000 rpm and the time is 10~30 min.

8. The method for preparing the nanozyme M-SA / NC@CDs as described in claim 3, characterized in that: In step (3), the molecular weight cutoff of the dialysis bag is 500 Da to 3000 Da, and the total dialysis time is 24 to 72 hours.

9. The use of the nanozyme M-SA / NC@CDs as described in claim 1 or 2 in the preparation of a drug for treating fungal keratitis.

10. An antifungal eye drop comprising the nanozyme M-SA / NC@CDs as described in claim 1 or 2, characterized in that: The antifungal eye drops are composed of nanozyme M-SA / NC@CDs and water for injection, and the mass-to-volume ratio of nanozyme M-SA / NC@CDs to water for injection is at least 7.8 μg / mL.

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