Ruthenium-based nano-enzyme with eye infiltration capacity as well as preparation method and application of ruthenium-based nano-enzyme

Abstract

The invention discloses a ruthenium-based nano-enzyme with eye infiltration capacity and a preparation method and application thereof, and relates to the technical field of medicine preparation, the ruthenium-based nano-enzyme is composed of a nano-enzyme matrix and fluorinated lipidosome wrapping the nano-enzyme matrix, and the nano-enzyme matrix is PolyRu with similar superoxide dismutase and catalase activity. According to the invention, the PolyRu nano-enzyme is encapsulated in the fluorinated liposome, so that the lipophilicity of the fluorinated liposome is enhanced, the fluorinated liposome can effectively penetrate through a cell membrane, the permeability of a medicine is further improved, and the non-invasive eye drops can be used for treating RND (Reduced Newcastle Disease); the technical problem that the existing nano-enzyme difficultly drips into eyeballs to effectively enter the eyeballs and is fully accumulated in the retina area to treat RND is solved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug preparation, and in particular to a ruthenium-based nanozyme with eye penetration ability, a preparation method and application thereof. Background Art

[0002] Retinal neovascularization disease (RND) is a series of eye diseases characterized by pathological proliferation of retinal blood vessels, which seriously threatens vision. Such diseases include diabetic retinopathy, retinopathy of prematurity and wet age-related macular degeneration. Their common feature is the abnormal growth of fragile and easily ruptured blood vessels in the retina or subretinal tissue, leading to complications such as retinal edema, hemorrhage, accumulation of exudates and retinal detachment, thereby impairing the patient's vision.

[0003] Currently, the treatment of RND mainly focuses on anti-angiogenic therapy, especially anti-vascular endothelial growth factor (anti-VEGF) therapy. Although these drugs can effectively inhibit abnormal vascular growth and improve related symptoms, they require frequent intraocular injections, which not only imposes a heavy economic and psychological burden on patients, but may also lead to drug resistance problems, thereby reducing efficacy. Therefore, existing treatments face the dual challenges of low patient compliance and reduced efficacy, and a new treatment strategy needs to be developed.

[0004] Studies have shown that nanozymes, as a new type of nanomaterial, can provide an effective alternative. Nanozymes have demonstrated strong antioxidant capacity in multiple biomedical fields such as oncology, gastroenterology and neurology. They can simulate the activity of natural enzymes such as superoxide dismutase (SOD) and catalase (CAT) to effectively remove ROS. In addition, nanozymes have stronger stability and superior bioavailability, can function for a long time in complex biological environments, and their nanoscale properties also help to penetrate general biological barriers. Therefore, it is expected that drugs can be made into nanozyme eye drops to treat eye diseases. For example, Dr. Li Jingguo's research team cooperated with Harvard University and other research teams to publish the article "Dual-Atom Nanozyme Eye Drops Attenuate Inflammation and Break the Vicious Cycle in Dry Eye Disease" in the internationally renowned academic journal Nano-Micro Letters, which disclosed the research results of using nanozyme eye drops to reduce inflammation, break the vicious cycle of dry eye, and effectively treat dry eye. However, in the actual treatment of retinal neovascular disease, we found that ordinary nanozymes are difficult to effectively enter the eyeball and accumulate sufficiently in the retinal area to treat RND. Therefore, further research on nanozymes is needed to make the drug into nanozyme eye drops to treat RND. Summary of the invention

[0005] The present invention provides a ruthenium-based nanozyme with eye penetration ability. The ruthenium-based nanozyme is a PolyRu nanozyme with SOD and CAT activities encapsulated in a fluorinated liposome, so that the nanozyme can effectively enter the inside of the eyeball and fully accumulate in the retinal area to treat RND, thereby making it possible to treat RND with non-invasive eye drops, solving the technical problem that existing nanozymes are difficult to effectively enter the inside of the eyeball by dropping into the eyeball and fully accumulate in the retinal area to treat RND.

[0006] The technical solution adopted by the present invention is as follows:

[0007] A ruthenium-based nanozyme with eye penetration ability, the ruthenium-based nanozyme consists of a nanozyme matrix and a fluorinated liposome encapsulating the nanozyme matrix, and the nanozyme matrix is ​​PolyRu with superoxide dismutase and catalase-like activities.

[0008] Furthermore, the fluorinated liposome is also modified with RGD.

[0009] Furthermore, the preparation process of the nanozyme matrix PolyRu includes the following steps:

[0010] (1) dissolving polyvinyl pyrrolidone in methanol to obtain solution A;

[0011] (2) Add Ru to solution A 3+ deionized water solution of salt, and react with magnetic stirring at 60-80°C for 3 hours;

[0012] (3) After the reaction is completed, the reaction mixture is centrifuged to remove excess methanol, and then subjected to ultrasonic treatment;

[0013] (4) Add an equal amount of chloroform and five times the volume of n-hexane to the residue obtained after ultrasonic treatment, wash the residue multiple times until the pH value of the residue reaches neutral, and then dry the washed material in a vacuum oven to obtain PolyRu.

[0014] The present invention also provides a method for preparing a ruthenium-based nanozyme having eye penetration ability, comprising the following steps:

[0015] (1) dissolving the liposome raw material in an organic solvent to form a liposome mixture;

[0016] (2) After the liposome mixture is spun into a film, PolyRu is added;

[0017] (3) After ultrasonic mixing for 5 minutes, the ruthenium-based nanozyme with eye penetration ability can be obtained.

[0018] Preferably, the organic solvent is a mixed solvent of methanol and chloroform in a volume ratio of 1:2.

[0019] More preferably, the liposome raw material consists of DSPE-PEG-F7, lecithin and cholesterol, and the obtained ruthenium-based nanozyme with eye penetration ability is F-PolyRu nanozyme.

[0020] More preferably, the liposome raw material consists of DSPE-PEG-F7, DSPE-PEG-RGD, lecithin and cholesterol, and the obtained ruthenium-based nanozyme with eye penetration ability is FR-PolyRu nanozyme.

[0021] According to the tissue penetration ability of the obtained ruthenium-based nanozyme, the present invention applies the obtained ruthenium-based nanozyme to the preparation of a drug for treating retinal neovascularization disease.

[0022] According to the tissue penetration ability of the obtained ruthenium-based nanozyme, the present invention also applies the obtained ruthenium-based nanozyme to the preparation of eye drops for treating retinal neovascularization diseases.

[0023] In summary, compared with the prior art, the present invention has the following advantages and beneficial effects:

[0024] 1. The present invention encapsulates PolyRu nanozymes in fluorinated liposomes to enhance their lipophilicity, enabling them to effectively penetrate cell membranes, thereby improving the permeability of drugs, making it possible to treat RND with non-invasive eye drops, thus solving the technical problem that existing nanozymes are difficult to effectively enter the eyeball by dropping into the eyeball and fully accumulate in the retinal area to treat RND;

[0025] 2. The present invention also achieves targeted delivery to retinal vascular lesions through the modification of RGD, further improving the therapeutic effect of ruthenium-based nanozymes on RND;

[0026] 3. The ruthenium-based nanozyme prepared by the present invention exhibits SOD and CAT cascade catalytic ability, can efficiently remove ROS, and inhibit cell damage caused by oxidative stress. This antioxidant mechanism not only helps to alleviate the oxidative imbalance in retinal neovascular diseases, but also reduces abnormal angiogenesis. The obtained ruthenium-based nanozyme significantly inhibits abnormal angiogenesis in the OIR model and repairs the avascular area of ​​the retina, showing a strong retinal vascular protection and repair function, and is expected to become an effective means of treating neovascularization-related eye diseases;

[0027] 4. The ruthenium-based nanozymes provided by the present invention show high biosafety, including no obvious adverse effects on body weight, blood parameters, liver and kidney function and tissue structure, ensuring its potential for long-term application;

[0028] 5. The present invention found through transcriptomics and protein level analysis that the FR-PolyRu nanozyme eye drops provided can regulate key signaling pathways such as PI3K-AKT, HIF-1 and MAPK, slowing down cell apoptosis and abnormal angiogenesis caused by hypoxia. It restores retinal homeostasis by regulating the expression of related genes (such as Flt4, Vegfa, etc.) and proteins (such as ERK, PI3K and AKT). BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Figure 1 is a characterization diagram of the physicochemical characteristics of FR-PolyRu nanozyme. A is a schematic diagram of the synthesis route of FR-PolyRu nanozyme. B is a TEM characterization result diagram of PolyRu, scale = 100nm. C is a TEM characterization result diagram of FR-PolyRu nanozyme, scale = 100nm. DE is an AFM topological image of FR-PolyRu nanozyme, with a scanning area of ​​1μm×1μm, D is a three-dimensional rendering of the topological image in the panel, and E is a corresponding height distribution diagram along the nanozyme in the panel, scale = 200nm. F is a dispersion diagram of FR-PolyRu nanozyme of different concentrations in deionized water. G is an elemental analysis diagram of FR-PolyRu nanozyme by XPS. H is a C1s+Ru3d high-resolution XPS spectrum of FR-PolyRu nanozyme. I is a particle size distribution diagram of RuO2, PolyRu, and FR-PolyRu nanozyme measured by DLS. J is a potential diagram of RuO2, PolyRu, and FR-PolyRu nanozyme measured by DLS. K is the dispersion diagram of RuO2, PolyRu and FR-PolyRu nanozymes in deionized water on the 0th, 7th, 14th and 30th days.

[0030] Figure 2 Characterization diagram of DSPE-PEG-NH2 (black) and DSPE-PEG-F7 (red) using 1H NMR (400 MHz, D2O).

[0031] Figure 3 The particle size distribution of RuO2, PolyRu and FR-PolyRu nanozymes measured by DLS. A is the particle size distribution of RuO2 dispersed in pH 7.4 solution for 1, 7, 14 and 30 days. B is the particle size distribution of PolyRu dispersed in pH 7.4 solution for 1, 7, 14 and 30 days. C is the particle size distribution of FR-PolyRu nanozyme dispersed in pH 7.4 solution for 1, 7, 14 and 30 days.

[0032] Figure 4Figure 2 is the zeta potential diagram of RuO2, PolyRu and FR-PolyRu nanozymes measured by DLS. A is the zeta potential diagram of RuO2 dispersed in pH 7.4 solution for 1, 7, 14 and 30 days. B is the zeta potential diagram of PolyRu dispersed in pH 7.4 solution for 1, 7, 14 and 30 days. C is the zeta potential diagram of FR-PolyRu nanozyme dispersed in pH 7.4 solution for 1, 7, 14 and 30 days.

[0033] Figure 5 Figure 1 is a diagram of the enzyme-like activity detection and theoretical analysis of FR-PolyRu nanozyme. A is a schematic diagram of the multi-enzyme activity of FR-PolyRu nanozyme. B is a diagram of the SOD-like activity of RuO2, PolyRu and FR-PolyRu nanozymes detected by the xanthine oxidase / cytochrome C system under pH 7.4 (n=3). C is a diagram of the catalase-like activity of nanozymes detected by recording the amount of dissolved oxygen produced in the H2O2 solution containing RuO2, PolyRu and FR-PolyRu nanozymes under pH 7.4 (n=3). D is a diagram showing that FR-PolyRu nanozyme can reduce the generation of superoxide radicals through EPR spectroscopy. E is a diagram showing that FR-PolyRu nanozyme can reduce the generation of singlet oxygen through EPR spectroscopy. F is a diagram showing that FR-PolyRu nanozyme can reduce the generation of hydroxyl radicals through EPR spectroscopy. G is a diagram of the ABTS free radical scavenging rate of RuO2, PolyRu and FR-PolyRu nanozymes (n=3). H is the DPPH radical scavenging rate diagram of RuO2, PolyRu and FR-PolyRu nanozymes (n=3). I is the side view and top view of the structural model of PolyRu nanozyme. JK is the schematic diagram of the SOD-like (J) and CAT-like (K) activity mechanism of PolyRu nanozyme. LM is the energy curve diagram (unit: eV) of the SOD-like (L) and CAT-like (M) mechanism of PolyRu nanozyme and the chemical composition diagram of each equilibrium point.

[0034] Figure 6 The decomposition diagram of H2O2 catalyzed by RuO2, PolyRu and FR-PolyRu nanozymes was detected by the change of absorbance at 240nm.

[0035] Figure 7 Figure 1 is a graph showing the multi-enzyme activity assay of nanozymes. Figure A is a graph showing the POD-like activity of RuO2, PolyRu and FR-PolyRu nanozymes (10 μg / mL) in the TMB-H2O2 system (n=3). Figure B is a graph showing the OXD-like activity of RuO2, PolyRu and FR-PolyRu nanozymes (10 μg / mL) in the TMB system (n=3).

[0036] Figure 8 This is the geometric structure diagram of the nanozyme-related intermediates.

[0037] Fig. 9 Figure 2 is a diagram of FR-PolyRu nanozyme clearing intracellular ROS and reversing oxidative damage in vitro. A is a schematic diagram of the in vitro antioxidant experiment of FR-PolyRu nanozyme. B is a diagram of the cell viability of HRECs cells co-cultured with different concentrations of RuO2, PolyRu and FR-PolyRu nanozymes for 24 hours detected by CCK-8 method. C is a diagram of the cell uptake of Cy5.5-FR-PolyRu observed by confocal laser scanning microscopy (CLSM), scale bar = 20μm. D is a diagram of the viability of HRECs cells co-cultured with 200μM H2O2 and different concentrations of RuO2, PolyRu, and FR-PolyRu nanozymes. (E) Representative immunofluorescence images showing RuO2, PolyRu and FR-PolyRu nanozymes stained with tubulin (red) and DAPI (blue) in treated HRECs cells. Scale bar = 20μm. FG is a flow cytometer test of ROS levels in HRCE cells stained with DCFH-DA and its statistical data (n = 3). HI is a representative immunofluorescence image and quantitative data of DCFH-DA (green) and DAPI (blue) staining in HRECs cells treated with RuO2, PolyRu or FR-PolyRu nanozymes. Scale bar = 200 μm (n = 3). JK is a flow cytometric test of mitochondrial membrane potential of HRECs cells treated with RuO2, PolyRu or FR-PolyRu nanozymes stained with JC-1 probe and its statistical data (n = 3). L is a graph of mitochondrial-specific ROS scavenging activity of RuO2, PolyRu or FR-PolyRu nanozymes, scale bar = 50 μm. M is a graph of cellular ATP levels of HRECs cells under different treatments (n = 3).

[0038] Fig.10Figure 2 shows the removal of intracellular ROS by FR-PolyRu nanozyme in HUVECs. A shows the cell viability of HUVECs cultured for 6 hours under different concentrations of H2O2 detected by CCK-8 method. B shows the cell uptake of Cy5.5-FR-PolyRu observed by confocal laser scanning microscopy (CLSM), scale bar = 20 μm. C shows the cell viability of HUVECs cultured for 6 hours under different concentrations of H2O2 detected by CCK-8 method. D shows the cell viability of HUVECs treated with 200 μM H2O2 and different concentrations of RuO2, PolyRu, and FR-PolyRu nanozymes. E shows a representative immunofluorescence image showing tubulin (red) and DAPI (blue) staining of HUVECs treated with RuO2, PolyRu, and FR-PolyRu nanozymes, scale bar = 20 μm. F is a flow cytometer detection of HUVECs ROS levels by DCFH-DA staining, G is a statistical data diagram (n = 3, one-way ANOVA and Tukey's multiple comparison test, **p < 0.01, ***p < 0.001). H is a representative immunofluorescence image of HUVECs cells treated with different concentrations of FR-PolyRu nanozymes, I is a quantitative data diagram of DCFH-DA (green) and DAPI (blue) staining, scale = 200 μm (n = 3, one-way ANOVA and Tukey's multiple comparison test, ***p < 0.001).

[0039] Fig.11 This is a graph showing the cell viability of HRECs cultured for 6 hours under the action of different concentrations of H2O2 detected by the CCK-8 method.

[0040] Fig.12 Representative immunofluorescence images of JC-1 aggregates (red) and JC-1 monomers (green) in HRECs cells treated with RuO2, PolyRu, and FR-PolyRu nanozymes. Scale bar = 50 μm.

[0041] Fig.13A is a diagram of FR-PolyRu nanozyme penetrating the ocular barrier. A is a schematic diagram of FR-PolyRu nanozyme penetrating the cornea to reach the fundus. B is an in vitro fluorescence imaging diagram that verifies the ability of ICG-labeled nanozyme to cross the cornea through the Transwell experiment. C is a quantitative analysis diagram of the average fluorescence intensity in the lower chamber after adding ICG-labeled nanozyme to the upper chamber at different time intervals. DE is a quantitative analysis diagram of the photoacoustic imaging of the intraocular nanozyme distribution at different time points after different nanozymes were instilled into the mouse eyeball and its corresponding average PA signal intensity (n=3). FG is an immunofluorescence staining image showing the Cy5.5 signal of the fundus after treatment with Free-Cy5.5 or Cy5.5-PolyRu nanozyme. Cy5.5 signal (red) and DAPI (blue), scale bar = 200μm, and its corresponding quantitative analysis diagram of fluorescence intensity (n=3).

[0042] Fig.14 Figure 2 shows that PolyRu nanozyme effectively reduces abnormal neovascularization through intraocular injection. A is a schematic diagram of the OIR mouse model. B is a schematic diagram of the in vivo antioxidant treatment experiment in OIR mice. C is a representative image of the whole retinal flat mount samples after treatment in different groups. Purple: avascular area, red: neovascular area. Scale bar = 1000 μm. D is a quantitative analysis of the percentage of avascular area. E is a quantitative analysis of the percentage of neovascular area (n = 3, one-way ANOVA and Tukey's multiple comparison test, *p < 0.05, **p < 0.01).

[0043] Fig.15 Figure 2 shows the reversal of retinal vascular lesions in OIR mice by FR-PolyRu nanozyme eye drops. A is a schematic diagram of the OIR mouse model. B is a schematic diagram of the in vivo antioxidant therapy experiment in OIR mice. C is a representative image of the whole retinal flat mount samples after treatment in different groups. Purple: avascular area, red: neovascular area. Scale bar = 1000 μm. DE is a quantitative analysis of the percentage of avascular area (D) and neovascular area (E) (n = 3). F is a Tunel immunofluorescence staining image of the fundus after different treatments; Tunel (green) and DAPI (blue). Scale bar = 100 μm. G is a quantitative analysis of cell apoptosis after Tunel staining (n = 3).

[0044] Fig.16Figure 2 shows the dysregulated IGFBP6 / PI3K / ERK / AKT signaling pathway in the retina of OIR mice regulated by FR-PolyRu. A shows the PCA analysis of samples in different groups. B shows the KEGG pathway enrichment analysis of the OIR-PBS group compared with the normal group. The vertical and horizontal axes represent the pathway name and Rich factor, respectively. The size of the dots indicates the number of differentially expressed genes in the pathway, and the colors represent different q values. C shows the GSEA analysis of genes in apoptosis, HIF-1 signaling pathway, and PI3K-AKT signaling pathway (normal group vs. OIR-PBS group). D shows the GSEA analysis of genes in apoptosis, HIF-1 signaling pathway, and PI3K-AKT signaling pathway (OIR-PBS group vs. OIR-FR-PolyRu group). E shows the heat map of representative differentially expressed genes (DEGs) in different groups; red indicates upregulation, blue indicates downregulation; log2 fold change ≥1, q value <0.05. F is a quantitative graph of Igfbp6 mRNA expression level (n=3, one-way ANOVA and Tukey's multiple comparison test, ***p<0.001). G is a graph of the protein levels of Igfbp-6, ERK, p-ERK, PI3K, p-PI3K, AKT and p-AKT in the mouse retina detected by Western blotting, with β-actin as an internal reference. H is a quantitative statistical graph of Igfbp-6 protein, a graph of the ratio of p-ERK to total ERK, a graph of the ratio of p-PI3K to total PI3K, and a graph of the ratio of p-AKT to total AKT (n=3, one-way ANOVA and Tukey's multiple comparison test, *p<0.05, **p<0.01, ***p<0.001). I is a schematic diagram of FR-PolyRu nanozyme alleviating oxidative stress and reducing neovascularization.

[0045] Fig.17Figure 1 is a biosafety analysis diagram of FR-PolyRu. A is a schematic diagram of the in vivo safety evaluation experiment of PBS or FR-PolyRu nanozymes administered by eye drops. B is a weight monitoring diagram of healthy female C57 mice after continuous instillation of PBS or FR-PolyRu nanozymes (1 or 5 mg / mL) eye drops for 14 days (n=3). CD is a representative index diagram of liver function (AST, ALT) and kidney function (BUN, CREA) of normal mice after treatment with PBS or FR-PolyRu eye drops (n=3). E is a representative image of H&E staining of eyeballs and important organs of normal mice after treatment with PBS or FR-PolyRu eye drops for 14 days (n=3). F is a schematic diagram of the in vivo safety evaluation experiment of PBS or FR-PolyRu nanozymes administered by tail vein. G is a 14-day weight monitoring diagram of healthy female C57 mice after intravenous injection of PBS or FR-PolyRu nanozymes (7 or 35 mg / kg) (n=3). HI is a representative graph of liver function (AST, ALT) and kidney function (BUN, CREA) of normal mice after tail vein injection of PBS or FR-PolyRu (n=3). J is a representative graph of H&E staining of eyeballs and important organs of normal mice after intravenous injection of PBS or FR-PolyRu eye drops for 14 days (n=3).

[0046] Fig.18 Figure 1 shows blood routine parameters of healthy BALB / c mice after administration. A shows blood routine parameters after PBS or FR-PolyRu nanozyme (1 or 5 mg / mL) eye drops were instilled for 14 consecutive days, including WBC, RBC, HGB and PLT (n=3). B shows blood routine parameters detected in healthy female BALB / c mice 14 days after intravenous injection of PBS or FR-PolyRu nanozyme (7 or 35 mg / kg), including WBC, RBC, HGB and PLT (n=3). DETAILED DESCRIPTION

[0047] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0048] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0050] The present application will be described in detail below with reference to embodiments and experimental data.

[0051] Example 1

[0052] The synthesis process of PolyRu nanozyme, F-PolyRu nanozyme, R-PolyRu nanozyme, FR-PolyRu nanozyme and other related preparations, as well as the characterization process and results of the obtained nanozymes are as follows:

[0053] 1. Preparation of PolyRu Nanozyme

[0054] 133 mg of polyvinyl pyrrolidone (molecular weight: 30 kDa) was dissolved in 180 mL of methanol, followed by the addition of 20 mL of deionized RuCl3 solution (1 mg / mL). The reaction was stirred magnetically at 70 ° C and 400 rpm for 3 hours. After the reaction was completed, the mixture was centrifuged to remove excess methanol and subjected to ultrasonic treatment. After removing the methanol, the residue was collected in a centrifuge tube, an equal volume of chloroform and five times the volume of n-hexane were added, washed several times until the solution was neutral, and then dried in a vacuum oven.

[0055] 2. Preparation of DSPE-PEG-F7

[0056] 10 mg of perfluorobutyric anhydride and 50 mg of DSPE-PEG-NH2 were dissolved in 8 mL of methanol, and the resulting mixture was stirred at room temperature for 48 hours. The reaction mixture was then added to cold ether and washed three times with dichloromethane / methanol, and the precipitate was collected by centrifugation and dried.

[0057] 3. Preparation of F-PolyRu Nanozyme

[0058] 0.5 mg DSPE-PEG-F7, 7 mg lecithin, 2 mg cholesterol and 3 mL organic solvent (methanol: chloroform = 1:2) were added to a 100 mL flask, the solvent was evaporated to form a film, and then 2 mL PolyRu nanozyme solution (5 mg / mL) was added to wash the membrane, and finally the mixture was sonicated for 5 minutes.

[0059] 4. Preparation of R-PolyRu Nanozyme

[0060] 1 mg DSPE-PEG-RGD, 7 mg lecithin and 2 mg cholesterol were added to a 100 mL flask containing 3 mL of organic solvent (methanol: chloroform = 1:2), and the solvent was evaporated to form a membrane. Then, 2 mL PolyRu nanozyme (5 mg / mL) was added to wash the membrane, and the membrane was thoroughly mixed after ultrasonication for 5 minutes.

[0061] 5. Preparation of FR-PolyRu nanozyme: 0.5 mg DSPE-PEG-F7, 1 mg DSPE-PEG-RGD, 7 mg lecithin, 2 mg cholesterol and 3 mL organic solvent (methanol: chloroform = 1:2) were added to a 100 mL flask. After rotating to form a membrane, 2 mL PolyRu nanozyme solution (5 mg / mL) was added to wash the membrane, and the solution was ultrasonically mixed for 5 minutes. Cy5.5-FR-Poly Ru nanozyme was prepared in the same way, and 0.5 mg Cy5.5-DSPE-PEG was added.

[0062] 6. Characterization of Nanozymes

[0063] Leverage Thermo Scientific TM Talos TM The morphology of the nanozymes was analyzed in aqueous solution using an L120C transmission electron microscope (TEM) at an operating voltage of 120 kV. The morphology and height of the nanozymes were tested using an atomic force microscope (Bruker Dimension Icon, Germany). The crystal structure was analyzed using an X-ray diffraction (XRD) analyzer using a D8 Focus diffractometer (Bruker, Germany) using Cu Kα radiation. Evaluated. The elements and chemical bond information of the nanozyme were characterized by X-ray photoelectron spectroscopy (XPS) (ESCALab 250, Thermo Fisher Scientific, USA). 1H nuclear magnetic resonance (NMR) spectrum was obtained on a BrukerAvance III NMR spectrometer (400MHz), the solvent was D2O, and the chemical shift (ppm) used tetramethylsilane as an internal standard. Dynamic light scattering (DLS) and zeta potential tests were performed by a dynamic light scattering instrument (Nano-Zs90, Malvern, UK). The osmotic pressure of the nanozyme eye drops was tested using an ice point osmometer (Advanced, USA). The viscosity of the liquid was measured by a rotational viscometer (Brookfield DV-2Pro, USA).

[0064] Results: As shown in the figure Figure 1 As shown in A, PolyRu nanozyme is mixed with cholesterol, DSPE-PEG, DSPE-PEG-F7 and DSPE-PEG-RGD, and then encapsulated in liposomes after ultrasound to form FR-PolyRu nanozyme. The synthesis of ruthenium-based antioxidant PolyRu nanozyme is formed by mixing the polymer polyvinyl pyrrolidone (PVP) with RuCl3. Subsequently, the amine group of DSPE-PEG is fluorinated with perfluorobutyric anhydride to synthesize DSPE-PEG-F7, and then1 H NMR spectroscopy ( Figure 2 ) was confirmed. Transmission electron microscopy (TEM) images showed that PolyRu nanozymes are spherical structures with a diameter of about 30nm ( Figure 1 B). Negative staining TEM images further confirmed that the liposomes were effectively encapsulated on the surface of PolyRu nanozymes ( Figure 1 C). At the same time, atomic force microscopy (AFM) showed that FR-PolyRu nanozyme has an irregular spherical structure with a diameter of about 30nm and a height of about 7nm ( Figure 1 D-1E). Clarity is very important for eye drops application, such as Figure 1 As shown in F, the nanozyme solution showed high clarity.

[0065] The effectiveness and stability of eye drops are crucial to the user experience, so a systematic evaluation was also conducted on the physicochemical properties of FR-PolyRu nanozyme eye drops, including osmotic pressure, torque, viscosity, shear stress, and shear rate. Table 1 shows that the basic properties of FR-PolyRu nanozyme eye drops are similar to the formula of the commercial brand Bausch + Lomb, indicating that it is suitable for ocular use. The structure of FR-PolyRu nanozyme was further analyzed by X-ray photoelectron spectroscopy (XPS). Figure 1 The XPS scanning spectrum of G confirms the presence of O, C, N and Ru, indicating that Ru has been successfully incorporated into the material. High-resolution Ru 3d XPS spectrum ( Figure 1 H) reveals that the binding energies of Ru 3d 5 / 2 and Ru 3d 3 / 2 are 280.47 eV and 284.64 eV, respectively, which is consistent with metallic Ru. In addition, the peaks at 281.11 eV and 285.20 eV are attributed to Ru 4+ 3d 5 / 2 and Ru 4+ 3d 3 / 2. These binding energies indicate that electron transfer can occur between Ru and the substrate, making Ru in an electron-rich state, thus showing strong reduction performance. This electron-rich state makes it difficult for adsorbed oxidizing substances to stabilize, thereby balancing the adsorption and desorption of reaction intermediates and promoting the reaction. Computational studies can further verify this inference.

[0066] Table 1 Basic parameters of FR-PolyRu nanozyme eye drops and commercial eye drops

[0067]

[0068] Stability is extremely critical during the development of eye drops, as maintaining the stability of the nanozyme formulation during storage is essential for maintaining efficacy. To this end, we studied the changes in particle size, zeta potential, and dispersibility of RuO2, PolyRu, and FR-PolyRu nanozymes over 30 days. Figure 1 As shown in Figures 1 and 1J, the initial particle size of RuO2, PolyRu, and FR-PolyRu nanozymes was approximately 50 nm. Both RuO2 and FR-PolyRu nanozymes exhibited positive zeta potential, which facilitated their retention in the eye and increased permeability through the negatively charged ocular surface. Over a 30-day period, the particle size of RuO2 and PolyRu nanozymes increased significantly, reaching approximately 200 nm and 100 nm, respectively, with a decrease in dispersibility. In contrast, FR-PolyRu nanozymes maintained a stable particle size and dispersibility throughout the study period ( Figure 1 K and Figure 3 ). In addition, the zeta potentials of all three nanozymes were relatively stable ( Figure 4 ). These findings indicate that FR-PolyRu nanozymes possess superior stability in eye drops applications, especially in terms of particle size, zeta potential, and dispersibility.

[0069] Example 2

[0070] In this example, the SOD and CAT cascade catalytic activities of the obtained nanozymes were detected and analyzed, including the following operations:

[0071] 1. Superoxide dismutase (SOD)-like activity assay

[0072] The SOD-like activity level of nanozymes was determined by measuring their response to O2 ·- to determine the clearance rate.

[0073] The principle of the method is as follows: O2 is produced in the xanthine-xanthine oxidase system ·- , O2 ·- It can reduce partially oxidized cytochrome C to reduced cytochrome C, which has maximum light absorption at 550nm. ·- The reduction of cytochrome C is inhibited, so the O2 ·-The inhibition rate was used to indirectly detect the SOD-like activity of the nanozyme. The total reaction system was 300 μL: including 50 μL xanthine, 50 μL cytochrome C, 20 μL xanthine oxidase and 180 μL PBS. The absorbance increase ΔA1 of cytochrome C for 1 minute was measured at 550 nm by a UV spectrophotometer. Keeping the total amount unchanged, adjust the volume of xanthine oxidase and PBS to make ΔA1 reach 0.0225, and then add the nanozyme to measure the absorbance increase ΔA2 of cytochrome C at 550 nm. O2 ·- The clearance rate was calculated according to the following formula: (ΔA1-ΔA2) / ΔA1×100%.

[0074] 2. Catalase (CAT)-like activity assay

[0075] A dissolved oxygen meter (JPBJ-608, Leici) was used to detect the dissolved oxygen concentration (10 μL / mL) of different nanozymes in H2O2 aqueous solution (200 mM, pH 7.4) to evaluate the CAT-like activity of the nanozymes.

[0076] H2O2 decomposition experiment: Different nanozymes (10 μg / mL) were mixed with 3 mL of H2O2 solution (10 mM, pH 7.4) in a cuvette, and the absorbance of the reaction system was continuously monitored at 240 nm for 10 minutes by a UV spectrophotometer to compare the ability of different nanozymes to degrade H2O2.

[0077] 3. Determination of peroxidase-like activity and oxidase-like activity

[0078] The peroxidase-like activity and oxidase-like activity of the nanozyme were evaluated by measuring the absorbance of TMB at 652 nm for 10 minutes using a microplate reader. The reaction system for peroxidase-like activity detection included 10 μL nanozyme (100 μg / mL, 50 mM phosphate buffer, pH 7.4), 1 μL TMB (20 mg / mL in DMSO), 5 μL H2O2 (10 M), and 84 μL PBS. The oxidase-like activity reaction system included 0 μL nanozyme (100 μg / mL, 50 mM phosphate buffer, pH 7.4), 1 μL TMB (DMSO 20 mg / mL), and 89 μL PBS.

[0079] 4. Nanozyme-induced O2 ·- , 1 ESR measurement of O2 and ·OH generation

[0080] Electron spin resonance (ESR) spectroscopy was performed at room temperature using a Bruker electron spin resonance (ESR) spectrometer (A300-10 / 12, Germany). DMPO was used as the O2 ·- and ·TEMP was used as a scavenger for OH 1 O2 capture agent.

[0081] 5. DPPH free radical scavenging experiment

[0082] Different concentrations of RuO2, PolyRu and FR-PolyRu were added to an ethanol solution containing 2,2-diphenyl-1-picrylhydrazyl (DPPH). After mixing, the final concentration of DPPH was 62.5 μM, and the final concentration of nanozyme was 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, 200 μg / mL. After 30 minutes of reaction, the absorbance of DPPH at 517 nm was detected. The calculation formula of DPPH free radical scavenging activity is as follows:

[0083] DPPH radical cation scavenging activity = [A DPPH -(A 样品 -A 空白 )] / A DPPH ×100%

[0084] Where ADPPH is the absorbance of DPPH solution, A 样品 is the absorbance of the sample after reaction with DPPH, A 空白 is the absorbance of the blank solution.

[0085] 6. ABTS free radical scavenging experiment

[0086] The free radical scavenging activity of 2,2'-diamino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) was evaluated by the ABTS free radical scavenging assay kit (Solarbio, BC4770). The experiment was performed according to the instructions. The final concentrations of the nanozyme were 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg / mL. The ABTS free radical scavenging activity was calculated as follows:

[0087] ABTS free radical cation scavenging activity = [A ABTS -(A 样品 -A 空白 ))] / A ABTS ×100%

[0088] Where AABTS is the absorbance of ABTS solution, A 样品 is the absorbance of the sample after reaction with ABTS, A 空白 is the absorbance of the blank solution.

[0089] 7. Spin-polarized density functional theory (DFT) calculations were performed using the Vienna Ab-initio Simulation Package (VASP) to study the adsorption properties of the synthesized materials. The projected augmented wave (PAW) method was employed with a cutoff energy of 450 eV and the Perdew-Burke-Ernzerhof (PBE) functional. In order to take into account van der Waals interactions, the DFT-D3 method was applied. Amorphous ruthenium (a-Ru) was constructed using ab initio molecular dynamics (AIMD) simulations. The optimized Ru crystals were heated from 500 K to 3000 K with a velocity scaling of 1.25 ps every 20 MD steps and then equilibrated at 2000 K for 0.5 ps with a time step of 1 fs to obtain the a-Ru structure. Subsequently, a PVP molecule containing three units was placed on the surface to create the a-Ru-PVP model. All models were fully relaxed, and the convergence criteria for energy and force were set to 10, respectively. -5 eV and The K-point grid uses Gamma points, and the adsorption energy (Eads) is calculated according to Eq. 1.

[0090] E ads =E total -E substrate -E adsorbate (1)

[0091] Etotal, Esubstrate, and Eadsorbate represent the energies of adsorption structure, substrate, and adsorbate, respectively. The free energy was calculated using the following formula 2.

[0092] G=E DFT +ZPE-TS (2)

[0093] where G, EDFT, ZPE and TS represent free energy, DFT-calculated energy, zero-point energy and entropy contribution, respectively.

[0094] Results: The SOD / CAT cascade catalytic activity of FR-PolyRu nanozymes can effectively catalyze cytotoxic O2 ·- Converted to O2, thereby reducing ROS levels ( Figure 5 A). Compared with the RuO2 control group, PolyRu and FR-PolyRu nanozymes showed higher SOD-like activity, while liposome encapsulation did not affect the O2 ·- Cleaning ability ( Figure 5 B). In addition, the excellent CAT-like activity of FR-PolyRu nanozyme was further confirmed by the measurement of H2O2 decomposition and O2 generation ( Figure 5 C and Figure 6). At physiological pH 7.4, FR-PolyRu nanozyme showed no peroxidase (POD) or oxidase (OXD)-like activity, indicating its high efficiency in selectively scavenging ROS ( Figure 7 ). Electron spin resonance (ESR) analysis supported the ROS scavenging ability of FR-PolyRu nanozymes, which significantly reduced O2 ·- , 1 O2 and OH content ( Figure 5 D-5F). DPPH and ABTS free radical inhibition assays further verified the superior free radical scavenging ability of FR-PolyRu nanozyme compared with the RuO2 control.

[0095] To elucidate the SOD and CAT cascade activities of PolyRu nanozymes, density functional theory (DFT) calculations were performed. The dynamic equilibrium structure of PolyRu (with one PVP molecule adsorbed on the surface) simulated by ab initio molecular dynamics (AIMD) was used as the initial model ( Figure 5 I) to study the catalytic mechanism. Figure 5 J and 5K present the proposed mechanisms for the SOD- and CAT-like activities of PolyRu nanozymes based on previous studies. The corresponding Gibbs free energy distributions are shown in Figure 5 L and 5M, and structural details of the intermediate species are shown in Figure 8 .

[0096] Gibbs free energy distribution showed that PolyRu nanozyme supports the occurrence of SOD and CAT-like enzyme cascade reactions, ultimately catalyzing O2 ·- (or ·OOH in acidic conditions) into O2. As an active reducing metal, ruthenium is the key reactant that enables PolyRu nanozymes to effectively adsorb SOD and CAT reactions, especially ·OOH and H2O2, thereby promoting subsequent chemical transformations. For example, Figure 5 In the second step of L, ·OOH is easily adsorbed on the surface of PolyRu, capturing hydrogen atoms from H2O* (indicates the adsorption state) to generate H2O2. This process is spontaneous and the reaction energy is -1.37 eV. Similarly, Figure 5The first step of M shows that H2O2 decomposes directly into O and H2O after adsorption on the PolyRu surface, with a release energy of -4.24 eV. The rate-limiting step in both the SOD and CAT catalytic cycles involves oxygen release, with a desorption energy of 1.63 eV, indicating that the PolyRu surface has a strong affinity for reactive oxygen species. However, this did not hinder the reaction cycle, because the competitive adsorption of ·OOH and H2O2 was sufficient to replace the surface-bound O2 for the next cycle, with adsorption energies reaching -1.62 eV (·OOH and H2O co-adsorbed) and -4.24 eV, respectively. These results provide valuable insights into the molecular mechanisms of the SOD- and CAT-like cascade activities of PolyRu nanozymes.

[0097] Example 3

[0098] In this example, the antioxidant and oxidative damage protection capabilities of the obtained nanozymes on HRECs and HUVECs were detected and analyzed, including the following operations:

[0099] 1. Cell culture

[0100] Human umbilical vein endothelial cells (HUVECs) and human corneal epithelial cells (HCECs) were purchased from the American Type Culture Collection (ATCC). HUVEC cell lines were cultured in RPMI-1640 medium (Gibco) containing 10% fetal bovine serum (FBS) (VivaCell, C04001) and 1% penicillin / streptomycin (Gibco). HCECs cell lines were cultured in Eagle's minimum essential medium (EMEM) (BNCC, 360906) containing 10% FBS (VivaCell, C04001) and 1% penicillin / streptomycin (Gibco). Human retinal endothelial cells (HRECs) (Seattle, WA, USA) were cultured in EGM-2 medium (Lonza, CC-4176). All three cell lines were cultured in a cell culture incubator at 37°C and 5% CO2.

[0101] 2. In vitro cytotoxicity

[0102] HRECs or HUVECs cells were seeded in a 96-well culture plate at a density of 5000 cells / well and cultured in an incubator for 24 hours, then different concentrations (0-100 μg / mL) of RuO2, PolyRu and FR-PolyRu nanozymes were added and cultured for another 24 hours. The viability of cells after treatment with different materials was detected by CCK8 assay.

[0103] 3. Cellular uptake experiment

[0104] HRECs or HUVECs cells were seeded in 12-well plates and cultured in confocal culture dishes for 24 hours. Cy5.5-labeled FR-PolyRu nanozymes were added to the confocal culture dishes and incubated for 1, 2, 4 or 8 hours, respectively. The uptake of nanozymes by cells was observed by confocal microscopy, with DAPI-stained cell nuclei in blue and Cy5.5-labeled FR-PolyRu in green.

[0105] 4. Cell Viability Restoration Experiment

[0106] HRECs or HUVECs cells were seeded on 96-well plates at a density of 10,000 cells / well and cultured in an incubator for 24 hours. Then, different concentrations of H2O2 (0-400 μM) were added and incubated for 6 hours. Finally, the toxicity of H2O2 on cells was detected by CCK-8 assay. HRECs or HUVECs cells were seeded on 96-well plates at a density of 6,000 cells / well and cultured in an incubator for 24 hours. Then, different concentrations of nanozymes (25, 50, 100 μg / mL) were added to pretreat the cells for 18 hours. Then, the old culture medium was replaced with a new culture medium containing 200 μM H2O2, and cultured for 6 hours with or without different concentrations of nanozymes, and finally, the cell viability was measured by CCK-8 assay.

[0107] 5. Tubulin Immunofluorescence Analysis

[0108] HRECs or HUVECs cells were seeded in confocal culture dishes and cultured for 24 hours. The experiment was divided into four groups, which were treated with new culture medium containing PBS, H2O2 (100μM), H2O2 (100μM) + PolyRu (100μg / mL) or H2O2 (100μM) + FR-PolyRu (100μg / mL), and incubated for another 6 hours. The cells were washed with PBS, fixed with 4% paraformaldehyde for 10 minutes, and then rinsed with PBS again. HUVECs cells were incubated with 0.1% TritonX-100 at room temperature for 10 minutes and then blocked with 5% BSA for 1 hour. Anti-tubulin antibody (Beyotime, AT819, 1:500) was added and incubated overnight at 4°C. The next day, the cells were incubated with fluorescent secondary antibody (Alexa 594) staining, and finally the cell nuclei were stained with DAPI, and the cell morphology was observed under a confocal microscope.

[0109] 6. Analysis of intracellular ROS levels

[0110] The fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) (Beyotime, S0033S) was used to detect the generation of intracellular ROS.

[0111] Flow cytometry (FITC channel) was used to detect changes in intracellular ROS: HRECs or HUVECs cells were seeded in 6-well plates, and the drug was added when the cell density reached about 70% and incubated for 6 hours. The cells were collected and incubated with DCFH-DA (10 μM) at 37 ° C in the dark for 30 minutes, and the changes in intracellular ROS were analyzed by flow cytometry. The experiment was divided into five groups: PBS, H2O2 (100 μM), H2O2 (100 μM) + RuO2 (100 μg / mL), H2O2 (100 μM) + PolyRu (100 μg / mL), H2O2 (100 μM) + FR-PolyRu (100 μg / mL).

[0112] Observation of ROS generation in HRECs cells by fluorescence microscopy: HRECs cells were seeded and cultured in 12-well plates. When the cell density reached about 70%, RuO2 (100 μg / mL), PolyRu (100 μg / mL) or FR-PolyRu (100 μg / mL) was added for 6 hours, followed by stimulation with 800 μM H2O2 for 30 minutes. Finally, DCFH-DA (10 μM) was incubated at 37°C in the dark for 30 minutes, and the cells were washed with PBS and photographed under a fluorescence microscope.

[0113] Observation of ROS generation in HUVECs cells by fluorescence microscopy: HUVECs cells were seeded and cultured in 12-well plates. When the cell density reached about 70%, FR-PolyRu nanozyme (25-100 μg / mL) was added and incubated for 6 hours, followed by stimulation with 800 μM H2O2 for 30 minutes. Finally, DCFH-DA (10 μM) was incubated at 37°C in the dark for 30 minutes, and the cells were washed with PBS and photographed under a fluorescence microscope.

[0114] 7. Mitochondrial membrane potential detection

[0115] The changes in mitochondrial membrane potential of HRECs cells were detected using a mitochondrial membrane potential detection kit (C2006, Beyotime Biotechnology) containing JC-1. HRECs cells were seeded in six-well plates or confocal culture dishes. When the cell density reached about 70%, the cells were incubated with RuO2 (100 μg / mL), PolyRu (100 μg / mL) or FR-PolyRu (100 μg / mL) for 6 hours and then stimulated with 800 μM H2O2 for 30 minutes. JC-1 and Hoechst 33342 were added to the confocal culture dish and incubated at 37°C for 20 minutes. After washing with PBS, the red channel of J-aggregates and the green channel of J-monomers were observed under a confocal microscope. In addition, the cells in the six-well plates were collected after drug incubation and incubated with JC-1 at 37°C for 20 minutes. After washing twice with PBS, the changes in mitochondrial membrane potential in the cells were observed by flow cytometry.

[0116] 8. MitoTracker and MitoSOX staining

[0117] To analyze the changes in mitochondrial membrane potential, HRECs cells were stained with MitoTracker Green (100nM, C1048, Beyotime Biotechnology); to analyze the generation of mitochondrial ROS, HRECs cells were stained with MitoSox Red (5μM, S0061S, Beyotime Biotechnology). HRECs cells were seeded in confocal culture dishes, and when the cell density reached about 70%, they were incubated with RuO2 (100μg / mL), PolyRu (100μg / mL) or FR-PolyRu (100μg / mL) for 6 hours, and then stimulated with 800μM H2O2 for 30 minutes. After replacing the new culture medium, MitoSox Red, MitoTracker Green and Hoechst 33342 (1:100, C1027, Beyotime Biotechnology) were added and incubated at 37°C in the dark for 30 minutes. After washing with PBS, images were taken under a confocal microscope.

[0118] 9. Intracellular ATP level detection

[0119] HRECs cells were seeded on six-well plates, and when the cell density reached about 70%, they were incubated with RuO2 (100 μg / mL), PolyRu (100 μg / mL) or FR-PolyRu (100 μg / mL) for 6 hours, and then stimulated with 800 μM H2O2 for 30 minutes. The ATP level of each group was then detected using an ATP assay kit (S0027, Beyotime Biotechnology). Specifically, the culture medium was removed, 200 μL of lysis solution was added and repeatedly pipetted. After cell lysis, centrifugation was performed at 12,000 rpm for 10 minutes, and the supernatant (20 μL) was mixed with the ATP detection working solution (100 μL) in an opaque 96-well plate. The luminescence intensity was monitored at 560 nm using a luminometer.

[0120] Results: To evaluate the antioxidant capacity of nanozymes in cells, we used the H2O2-induced oxidative damage model in HUVECs and HRECs. Subsequently, the ability of pre-treatment nanozymes to alleviate oxidative stress-induced cell damage was evaluated ( Fig. 9 A). To exclude the potential influence of nanomaterial toxicity on the evaluation of its protective ability, we initially treated cells with different concentrations of RuO2, PolyRu and FR-PolyRu nanozymes, and then tested cell viability by CCK8 method. The results showed that with increasing concentration, PolyRu and FR-PolyRu nanozymes had no significant effect on cell viability ( Fig. 9 B and Fig.10 A).

[0121] The cellular uptake efficiency of the nanozyme was further studied. The accumulation of FR-PolyRu nanozyme in cells gradually increased over time and reached a maximum value at 8 hours, indicating that it has a good cellular endocytosis effect ( Fig. 9 C and Fig.10 B). In addition, we observed that HRECs and HUVECs pretreated with PolyRu and FR-PolyRu nanozymes effectively alleviated the reduction in cell viability caused by 200 μM H2O2 ( Fig. 9 D, Fig.10 C-10D and Fig.11 ) and cytoskeletal disruption ( Fig. 9 E and Fig.10 E), which suggests that PolyRu nanozymes may scavenge ROS and alleviate cellular oxidative damage through their enzyme-like activity.

[0122] The effect of nanozymes on intracellular ROS levels was also evaluated. Fig. 9 F-9G and Fig.10As shown in F-10G, DCFH-DA was used to label intracellular ROS, and flow cytometry analysis showed that ROS increased significantly after H2O2 stimulation. However, compared with the H2O2 treatment group, the ROS level of cells pretreated with PolyRu and FR-PolyRu nanozymes was significantly reduced, close to that of the control group. Confocal imaging of intracellular DCFH-DA fluorescence staining ( Fig. 9 H-9I and Fig.10 These findings suggest that PolyRu and FR-PolyRu nanozymes alleviate cellular oxidative damage by reducing ROS levels.

[0123] Abnormally elevated intracellular ROS levels are often closely associated with mitochondrial dysfunction. Damaged mitochondria often lead to elevated ROS levels, which in turn trigger the destruction of intracellular lipids, proteins, and DNA, exacerbating mitochondrial damage. To determine whether PolyRu and FR-PolyRu nanozymes can alleviate mitochondrial damage, we conducted further evaluations. Using the JC-1 probe to measure mitochondrial membrane potential (MMP) under different conditions, we found that HRECs treated with H2O2 mainly showed green fluorescence, indicating that MMP was lower due to the formation of JC-1 monomers ( Fig. 9 J-9K and Fig.12 ). In contrast, cells treated with PolyRu and FR-PolyRu nanozymes maintained high MMP after H2O2 stimulation, as shown by red fluorescence of JC-1 aggregates. In addition, by quantifying mitochondrial ROS by MitoSOX probe, we observed a significant increase in mitochondrial ROS levels in H2O2-treated HRECs. However, cells treated with PolyRu and FR-PolyRu nanozymes maintained near-normal mitochondrial ROS levels ( Fig. 9 L-9M). These findings suggest that PolyRu and FR-PolyRu nanozymes alleviate mitochondrial oxidative damage by scavenging ROS in mitochondria, thereby maintaining mitochondrial membrane potential.

[0124] Example 4

[0125] This example detects and analyzes the tissue penetration ability of the obtained nanozyme, including the following operations:

[0126] 1. Experimental Animals

[0127] The C57BL / 6J mice used in the experiment were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China). All animal experiments were performed in accordance with the experimental protocols established by the Animal Ethics Committee of Zhengzhou University. All experimental animals were housed in a standard animal room with a light / dark cycle of 12 h / 12 ​​h. Ophthalmological examinations were performed on the mice before the experiment to exclude the possibility of eye diseases.

[0128] 2. Transwell experiment

[0129] HCECs cells were seeded in the upper chamber of Transwell at a density of 5×104 cells / well, and HRECs cells were seeded in the lower chamber at a density of 5×104 cells / well. After incubation for 24 hours, the drug was added to the upper chamber. The experiment was divided into five groups: Free-ICG, ICG-PolyRu, ICG-R-PolyRu, ICG-F-PolyRu, and ICG-FR-PolyRu. At 0, 12, and 48 hours after drug addition, the IVIS in vivo imaging system (LuminaXR series, Perkin Elmer) was used to take pictures to observe the permeation of the drug from the upper chamber to the lower chamber.

[0130] 3. In vivo photoacoustic tracking

[0131] Healthy C57BL / 6J mice were randomly divided into 5 groups (n=3): Free-ICG, ICG-PolyRu, ICG-R-PolyRu, ICG-F-PolyRu, and ICG-FR-PolyRu. The drug was topically administered via eye drops, 10 μL per mouse. After administration, the mice were placed in a dark environment. The photoacoustic signals in the eyes of the mice were detected at 0.5, 3, 4, 6, and 12 hours after administration. LAZR-X system for observation.

[0132] 4. Biodistribution and penetration of nanozymes in the eye

[0133] Healthy C57BL / 6J mice were randomly divided into three groups (n=3). Free-Cy5.5 and Cy5.5-PolyRu were administered topically by eye drops. The eyeballs were removed 1, 6, and 12 hours after administration, and quickly frozen in liquid nitrogen, embedded in OCT embedding medium, and 12 μm thick frozen sections were made and fixed on slides. The slides were washed three times with PBS and sealed with anti-fading mounting medium containing DAPI. The distribution of nanozymes in the eyeballs was observed using confocal microscopy.

[0134] Results: The penetration ability of eye drops in the eyeball is crucial to determine its accumulation concentration and efficacy in the fundus. To this end, we evaluated the penetration ability of FR-PolyRu eye drops in the mouse eyeball ( Fig.13 A) In the Transwell experiment, we densely seeded HCECs in the upper chamber and HRECs in the lower chamber to further evaluate the in vitro penetration ability of ICG-labeled FR-PolyRu nanozymes, specifically its ability to pass through the upper chamber. Fig.13As shown in B-13C, F-PolyRu and FR-PolyRu nanozymes successfully penetrated the HCECs in the upper chamber and migrated to the lower chamber, and their penetration degree gradually increased over time. In contrast, PolyRu and R-PolyRu nanozymes did not show such penetration ability, indicating that this ability is closely related to fluorination modification. In addition, we evaluated the permeability of FR-PolyRu nanozymes in mouse eyeballs. Photoacoustic imaging technology observed that PolyRu and R-PolyRu nanozymes mainly accumulated in the cornea, showing limited permeability. In contrast, F-PolyRu and FR-PolyRu nanozymes exhibited strong tissue penetration ability and were significantly enriched in the fundus area about 4 hours after administration ( Fig.13 D-13E). In addition, staining of frozen sections of mouse eyeballs showed that FR-PolyRu nanozymes can effectively accumulate in the retinal area of ​​the fundus ( Fig.13 F-13G). These results indicate that F-PolyRu and FR-PolyRu nanozyme eye drops have excellent tissue penetration ability, enabling them to effectively enter the interior of the eyeball and fully accumulate in the retinal area.

[0135] Example 5

[0136] This example detects and analyzes the effect of the obtained nanozyme in inhibiting retinal neovascularization in OIR mice, including the following operations:

[0137] 1. Establishment and treatment of oxygen-induced retinopathy (OIR) mouse model

[0138] Establishment of OIR animal model: Newborn mice and their mothers were placed in a hyperoxic feeding box (75% O2) for 5 days. Then, the 12-day-old mice and their mothers were transferred to normal air (21% O2) for 5 days. Finally, the mice were analyzed 17 days after birth.

[0139] In vivo treatment experiment (intraocular injection): OIR mice born on day 12 were randomly divided into groups and the drug was injected into the vitreous of the mice using a 30-gauge needle. The experimental groups included: untreated group, PBS group, RuO2 (1μg), PolyRu (0.5μg, 1μg, 2μg) group. Finally, the treatment effect of OIR mice was analyzed 17 days after birth.

[0140] In vivo treatment experiment (eye drops): OIR mice born on day 12 were randomly assigned and 2.5 μL of eye drops were instilled into each eye twice a day. The experiment was divided into untreated group, PBS group, RuO2 group, PolyRu group, F-PolyRu group and FR-PolyRu group. Finally, the treatment effect of OIR mice was analyzed 17 days after birth.

[0141] 2. Whole-retinal flat-mount immunostaining

[0142] The mouse eyeball was removed and fixed in 4% paraformaldehyde solution for 20 minutes. The retina was then dissected and divided into four petals. After dissection, the retina was washed three times with PBS (5 minutes each time) and then blocked at room temperature for 30 minutes in PBS containing 5% FBS. The tissue was purified overnight at 4°C with GS-IB4 homologous plant lectin antibody (I21411, Thermo Fischer, 1:200) and then washed three times with PBS (5 minutes each time). Then, the retina was flat-laid and a confocal microscope was used to obtain a whole retinal flat-mount image.

[0143] 3. TUNEL analysis

[0144] The TUNEL assay kit (Beyotime, C1086) was used to analyze the cell death of mouse retina after different treatments. In brief, frozen tissue sections of the eyeballs were fixed in 4% paraformaldehyde and washed twice with PBS. The slides were further incubated in PBS containing 0.5% Triton x-100 at room temperature for 5 minutes, and finally incubated with TUNEL assay solution for 60 minutes at 37°C in the dark. The slides were washed three times with PBS and mounted with anti-fading mounting medium containing DAPI. Cell apoptosis was observed using a confocal microscope.

[0145] 4. Transcriptome sequencing and data analysis

[0146] Mice were randomly selected from the normal group, PBS group (OIR) and FR-PolyRu group (OIR), and their retinal tissues were extracted (n=3). Total RNA was isolated from retinal tissues using TRIzol reagent (Solarbio, R1100) and then quickly frozen in liquid nitrogen. RNA sequencing analysis was performed by Biomarker Technologies (Beijing).

[0147] 5. RNA extraction and RT-qPCR analysis

[0148] Mice were randomly selected from the normal group, PBS group (OIR) and FR-PolyRu group (OIR), and their retinal tissues were extracted (n=3). Retinal RNA was extracted using TRIzol reagent (Solarbio, R1100) according to the manufacturer's instructions. Total RNA was reverse transcribed into complementary DNA (cDNA) according to the instructions of Hiscript III All-in-one RT SuperMix Perfect for qPCR Kit (R333-01, Vazyme). RT-qPCR analysis was performed according to the instructions of Taq Pro Universal SYBR qPCR Master Mix Kit (Q712-02, Vazyme). Relative mRNA expression was calculated by the comparative cycle threshold method (ΔΔCT method). All experiments were repeated three times. The primer sequences for amplification are listed in Table 2.

[0149] Table 2 Primer sequences

[0150]

[0151] 5. Western Blot Analysis

[0152] Mice were randomly selected from the normal group, PBS group (OIR) and FR-PolyRu group (OIR), and their retinal tissues were extracted (n=3). Total protein was extracted from mouse retinal tissue using a total protein extraction kit (BC3710, Solarbio), and the protein concentration was determined by BCA protein quantification (P0009, Beyotime Biotechnology). The extracted proteins were separated by SDS-PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with a specific primary antibody overnight at 4°C and then incubated with a specific secondary antibody for 1 hour at room temperature. The signal was detected using an ultrasensitive ECL chemiluminescence kit (P10018S, Beyotime Biotechnology). The primary and secondary antibodies used were as follows: rabbit anti-Igfbp6 (GB114043-100, Servicebio), rabbit anti-ERK (#4695T, Cell Signaling), rabbit anti-phospho-ERK (#4370T, Cell Signaling), rabbit anti-PI3K (ab191606, abcam), rabbit anti-phospho-PI3K (#4228T, Cell Signaling), rabbit anti-AKT (ab179463, abcam), rabbit anti-phospho-AKT (ab192623, abcam), mouse anti-β-actin (AF0003, Beyotime Biotechnology), anti-rabbit HRP-conjugated IgG (#7074, Cell Signaling), anti-mouse HRP-conjugated IgG (A0208, Beyotime Biotechnology). The primary antibody dilution ratio was 1:1000, and the secondary antibody dilution ratio was 1:5000.

[0153] Results: PolyRu and FR-PolyRu nanozymes exhibited antioxidant properties in cell models and were able to effectively reduce cellular oxidative damage. Therefore, we first evaluated the inhibitory effect of intravitreal injection of PolyRu nanozymes on abnormal retinal angiogenesis. Fig.14 As shown in A, 7-day-old mice were exposed to a high oxygen concentration environment (75%) for 5 days, which led to vascular degeneration and the formation of a central avascular zone. At P12, mice were transferred to a normal environment with 21% oxygen, which triggered retinal neovascularization due to relative hypoxia. We injected PolyRu nanozyme ( Fig.14 B) to evaluate its inhibitory effect on fundus neovascularization. Fig.14 C-14E ​​showed that PolyRu nanozyme treatment significantly reduced retinal neovascularization and central avascular zone in OIR mice, indicating that PolyRu nanozyme can promote retinal vascular repair.

[0154] Given the superior eye penetration ability of F-PolyRu and FR-PolyRu nanozymes, we further investigated the effects of FR-PolyRu nanozyme eye drops on abnormal retinal blood vessels in P12 mice ( Fig.15 A-15B). Fig.15 As shown in C-15E, both F-PolyRu and FR-PolyRu nanozyme eye drops can effectively inhibit abnormal retinal angiogenesis and accelerate the repair of the central avascular zone. FR-PolyRu may have a better effect based on the targeting effect of RGD; while PolyRu eye drops did not show this effect due to insufficient penetration. Further, we performed TUNEL staining analysis on retinal sections of OIR mice, and the results showed that the level of cell apoptosis in the retina of OIR mice was significantly higher than that of normal mice. It is worth noting that treatment with FR-PolyRu nanozyme eye drops effectively reversed the apoptosis of retinal cells ( Fig.15 F-15G). These results suggest that F-PolyRu and FR-PolyRu nanozymes can repair retinal vascular abnormalities non-invasively via eye drops, which is attributed to their tissue penetrating and antioxidant protective properties.

[0155] To explore the mechanism of action of retinal neovascularization at the transcriptional level, we performed transcriptome analysis of the mouse retina after nanozyme treatment by mRNA sequencing (mRNA-seq). The experimental groups included untreated retina (normal group), PBS-treated oxygen-induced retinopathy (OIR-PBS group), and FR-PolyRu eye drops-treated OIR group (OIR-FR-PolyRu group). Principal component analysis (PCA) showed that the FR-PolyRu treatment group had higher transcriptional similarity with the normal group compared with the OIR-PBS group, indicating that FR-PolyRu may play a protective role at the mRNA transcriptional level ( Fig.16 A). Pathway enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) showed that abnormal neovascularization in the OIR model mainly involved the PI3K-Akt, HIF-1, and MAPK signaling pathways ( Fig.16 B). Gene set enrichment analysis (GSEA) further showed that OIR treatment led to positive regulation of apoptosis, HIF signaling, and PI3K-AKT pathways, while FR-PolyRu treatment negatively regulated these pathways ( Fig.16C-16D). Given the significant impact of the PI3K-AKT pathway, we further analyzed the genes enriched in this pathway. The heat map of differentially expressed genes showed that angiogenesis-related downstream genes were significantly upregulated in the OIR group, including Flt4, Vegfa, Col1a2, Thbs1, and Pdgfd. FR-PolyRu treatment attenuated the abnormal expression of these genes, indicating its effectiveness in regulating the expression of angiogenesis-related genes ( Fig.16 E).

[0156] In addition to the above genes, heat map analysis also showed significant changes in the expression of insulin-like growth factor binding protein 6 (Igfbp6), which regulates the PI3K-AKT signaling pathway ( Fig.16 E). This regulation may trigger metabolic changes and oxidative stress bursts, indicating that Igfbp6 is closely related to the mitochondrial dysfunction mentioned above. Therefore, we further verified the expression of Igfbp6 at the transcriptional and protein levels and confirmed that it was significantly upregulated in the OIR group, while the expression level was reduced to near normal levels in the FR-PolyRu treatment group ( Fig.16 F-16H). To explore the downstream effects on the PI3K-AKT signaling pathway, we performed protein level verification. The results showed that the abnormal phosphorylation of ERK, PI3K, and AKT was significantly reduced after FR-PolyRu treatment, which is consistent with the observation that abnormal retinal neovascularization was improved ( Fig.16 G-16H). These findings suggest that FR-PolyRu effectively regulates the upstream and downstream elements of the PI3K-AKT signaling pathway, which may help restore retinal homeostasis ( Fig.16 I).

[0157] Example 6

[0158] This example tests and analyzes the biosafety of FR-PolyRu nanozyme, including the following operations:

[0159] 1. In vivo biosafety assessment

[0160] Healthy C57BL / 6J mice were randomly divided into groups (n=3) to evaluate the safety of FR-PolyRu nanozymes in mice after administration by eye drops or tail vein injection. Mice injected with tail vein were divided into three groups: PBS group, FR-PolyRu (7 mg / Kg) group and FR-PolyRu (35 mg / Kg) group. Mice administered with eye drops were divided into three groups: PBS group, FR-PolyRu (1 mg / mL) group and FR-PolyRu (5 mg / mL) group. Each mouse was administered 5 μL of eye drops once a day in each eye. After the start of administration, the status of the mice was continuously observed for 14 days, and the weight changes of the mice were recorded. On the 14th day, the mice were euthanized, blood was collected for blood biochemical testing, and the eyeballs, brain, heart, liver, spleen, lungs and kidneys were collected for H&E staining.

[0161] Results: To evaluate the biosafety of FR-PolyRu, mice were administered eye drops for 14 consecutive days, e.g. Fig.17 The results showed that the body weight of mice remained stable during the entire treatment period ( Fig.17 B). In addition, routine peripheral blood analysis on day 14 showed that FR-PolyRu eye drops had no adverse effects on white blood cells, red blood cells, hemoglobin, or platelets ( Fig.18 Blood biochemical tests further confirmed that liver and kidney function were not impaired by the treatment ( Fig.17 C-17D). In the H&E staining histological analysis 14 days later, there was no significant abnormality in the number and arrangement of retinal photoreceptor cells in mice treated with FR-PolyRu, and no significant pathological changes were observed in other organs ( Fig.17 E).

[0162] In addition, we further evaluated the toxicity of extremely high concentrations of FR-PolyRu. Cumulative doses of eye drops for 14 days were administered via a single tail vein injection and followed by observation for the next 14 days. Fig.17 G-17J and Fig.18 As shown in B, FR-PolyRu at this concentration did not cause any adverse effects on the body weight, blood parameters or organs of mice. The results showed that FR-PolyRu eye drops did not show obvious toxicity or adverse reactions in mice and had good biosafety.

[0163] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article, or device. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0164] The above-mentioned embodiments only express the specific implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the protection scope of the present application. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present application, and these all belong to the protection scope of the present application.

Claims

1. A ruthenium-based nanozyme with eye penetration ability, characterized in that: The ruthenium-based nanozyme consists of a nanozyme matrix and a fluorinated liposome encapsulating the nanozyme matrix. The nanozyme matrix is ​​PolyRu having superoxide dismutase and catalase-like activities.

2. The ruthenium-based nanozyme with eye penetration ability according to claim 1, characterized in that: The fluorinated liposome is further modified with RGD.

3. The ruthenium-based nanozyme with eye penetration ability according to claim 1, characterized in that: The preparation process of the nanozyme matrix PolyRu comprises the following steps: (1) dissolving polyvinyl pyrrolidone in methanol to obtain solution A; (2) Add Ru to solution A 3+ deionized water solution of salt, and react with magnetic stirring at 60-80°C for 3 hours; (3) After the reaction is completed, the reaction mixture is centrifuged to remove excess methanol, and then subjected to ultrasonic treatment; (4) Add an equal amount of chloroform and five times the volume of n-hexane to the residue obtained after ultrasonic treatment, wash the residue multiple times until the pH value of the residue reaches neutral, and then dry the washed material in a vacuum oven to obtain PolyRu.

4. The method for preparing a ruthenium-based nanozyme having eye penetration ability according to any one of claims 1 to 3, characterized in that: The steps include: (1) dissolving the liposome raw material in an organic solvent to form a liposome mixture; (2) After the liposome mixture is spun into a film, PolyRu is added; (3) After ultrasonic mixing for 5 minutes, the ruthenium-based nanozyme with eye penetration ability can be obtained.

5. The method for preparing a ruthenium-based nanozyme having eye penetration ability according to claim 4, characterized in that: The organic solvent is a mixed solvent of methanol and chloroform in a volume ratio of 1:

2.

6. The method for preparing the ruthenium-based nanozyme with eye penetration ability according to claim 5, characterized in that: The liposome raw material consists of DSPE-PEG-F7, lecithin and cholesterol, and the prepared ruthenium-based nanozyme with eye penetration ability is F-PolyRu nanozyme.

7. The method for preparing a ruthenium-based nanozyme having eye penetration ability according to claim 5, characterized in that: The liposome raw material consists of DSPE-PEG-F7, DSPE-PEG-RGD, lecithin and cholesterol, and the prepared ruthenium-based nanozyme with eye penetration ability is FR-PolyRu nanozyme.

8. Use of the ruthenium-based nanozyme with eye penetration ability as claimed in any one of claims 1 to 3 in the preparation of a drug for treating retinal neovascularization diseases.

9. Use of the ruthenium-based nanozyme with eye penetration ability as described in any one of claims 1 to 3 in the preparation of eye drops for treating retinal neovascularization diseases.

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