A zinc-doped ceria drug-loaded nanoscale enzyme platform, a preparation method and application thereof

Abstract

The application discloses a zinc-doped cerium oxide drug-loaded nanoscale enzyme platform and a preparation method and application thereof, and belongs to the technical field of biomedical nanomaterials. The nanoscale enzyme platform with a continuous functional interface is constructed through a specific preparation method: zinc-doped cerium oxide is used as an antioxidant core, a phenylboric acid modification layer is used as an ocular surface adhesion interface, and a mitochondrial fission inhibitor is used as a mitochondrial fission regulation drug, so that multi-level synergistic treatment of 'long-term residence on the ocular surface-ROS rapid removal-enhanced endogenous antioxidant-mitochondrial fission inhibition-reduced mtDNA leakage-inhibition of inflammasomes and pyroptosis' is finally realized. The design can more comprehensively intervene in the key pathological links of dry eye disease, and is suitable for constructing ocular surface drug preparation for treating dry eye disease.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, specifically to a zinc-doped cerium oxide drug-loaded nanozyme platform, its preparation method, and its applications. Background Technology

[0002] Dry eye disease (DED) is a chronic ocular surface disease caused by tear film homeostasis imbalance. Its development is closely related to ocular surface hyperosmolarity, inflammatory response, oxidative stress, mitochondrial damage, and corneal epithelial cell death. Dry eye-related stimuli can lead to a large accumulation of reactive oxygen species (ROS) in corneal epithelial cells. Excessive ROS further induces a decrease in mitochondrial membrane potential, Drp1-mediated excessive mitochondrial fission, and mitochondrial DNA leakage. Leaked mitochondrial DNA can promote the activation of NLRP3 and AIM2 inflammasomes and induce Caspase-1 / GSDMD-mediated pyroptosis, exacerbating ocular surface inflammation and corneal epithelial damage.

[0003] Current treatments for dry eye include artificial tears, anti-inflammatory drugs, immunosuppressants, and lacrimal secretion stimulants. However, these treatments typically suffer from short residence time on the ocular surface, limited target sites, and difficulty in simultaneously addressing oxidative stress and mitochondrial abnormalities.

[0004] Cerium oxide nanozymes have Ce 3+ / Ce 4+ Reversible redox cycles can mimic the activities of various antioxidant enzymes to scavenge reactive oxygen species. Phenylboronic acid modification can enhance ocular surface adhesion by binding to diol structures in ocular mucins or glycocalyxes. Zinc-doped cerium oxide can increase oxygen vacancies and improve antioxidant properties. However, in existing technologies, zinc-doped cerium oxide is mostly prepared using hydrothermal methods, co-precipitation methods, sol-gel methods, impregnation methods, or high-temperature solid-phase methods.

[0005] The aforementioned conventional metal doping methods typically suffer from problems such as harsh reaction conditions, the need for high-pressure or high-temperature calcination, easy particle aggregation, insufficient surface functionalizable sites, and poor suitability for subsequent drug loading and ocular surface delivery. In particular, existing technologies have not disclosed the preparation of zinc-doped cerium oxide nanozymes using a DMF / water dual-solvent system combined with a binary coordination competition system composed of 2-aminobenzimidazole and 2-methylimidazole, nor have they disclosed further grafting phenylboronic acid onto this material and loading Drp1-mediated mitochondrial fission inhibitors for cascade intervention in the ROS-mitochondrial fission-mtDNA-inflammation body-pyroptosis pathway in dry eye disease.

[0006] Therefore, there is still a need to develop a nanozyme therapy platform that has a mild preparation method, a continuously functionalizable structure, is suitable for ocular surface administration, and can simultaneously scavenge reactive oxygen species and inhibit abnormal mitochondrial division. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a zinc-doped cerium oxide drug-loaded nanozyme platform, its preparation method, and its applications, in order to improve drug residence time on the ocular surface, scavenge reactive oxygen species, inhibit Drp1-mediated excessive mitochondrial division, and block mtDNA-related inflammasome activation and pyroptosis.

[0008] The technical solution of the present invention to solve the above-mentioned technical problems is as follows: a zinc-doped cerium oxide drug-loaded nanozyme platform is provided, wherein the zinc-doped cerium oxide drug-loaded nanozyme platform uses zinc-doped cerium oxide nanozyme particles as an antioxidant core; the surface of the zinc-doped cerium oxide nanozyme particles has a phenylboronic acid modification layer as an ocular surface adhesion interface; the phenylboronic acid modification layer is loaded with mitochondrial division regulating drugs; the mitochondrial division regulating drugs include mitochondrial division inhibitors.

[0009] Furthermore, mitochondrial division regulating drugs also include at least one of anti-inflammatory drugs, repair-promoting drugs, and lubricating and protective molecules; wherein the anti-inflammatory drugs are at least one of dexamethasone, diclofenac sodium, cyclosporine A, and tacrolimus; the repair-promoting drugs are at least one of human epidermal growth factor and human fibroblast growth factor; and the lubricating and protective molecules are at least one of sodium hyaluronate and sodium carboxymethyl cellulose.

[0010] This invention provides a method for preparing the above-mentioned zinc-doped cerium oxide drug-loaded nanozyme platform, comprising the following steps: (1) Preparation of zinc-doped cerium oxide nanoparticles: A1. The first solution is prepared by mixing the cerium-containing precursor, the zinc-containing precursor and N,N-dimethylformamide. A2. Add 2-aminobenzimidazole, 2-methylimidazole and sodium formate to deionized water, mix well and prepare the second solution; A3. Mix the first solution from step A1 with the second solution from step A2, and react at 60℃-100℃ for 1-4 hours under stirring. A4. The product obtained in step A3 is centrifuged, washed and redispersed to obtain Zn-CeO2 nanoparticles. (2) Surface modification with phenylboronic acid: B1. Disperse the Zn-CeO2 nanoparticles prepared in step 1 in a buffer solution to obtain a Zn-CeO2 dispersion. B2. Dissolve phenylboronic acid or a carboxyl derivative of phenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in N,N-dimethylformamide to prepare a pre-activated mixture. B3. The pre-activated mixture from step B2 is added dropwise to the Zn-CeO2 dispersion from step B1. After stirring and reacting, the mixture is centrifuged, washed, and dried to obtain phenylboronic acid-modified zinc-doped cerium oxide nanoenzyme particles Zn-CeO2@P. (3) Preparation of zinc-doped cerium oxide drug-loaded nanozyme platform: C1. Zn-CeO2@P from step (2) is ultrasonically dispersed in purified water to obtain Zn-CeO2@P dispersion; C2. Dissolve the mitochondrial division inhibitor in dimethyl sulfoxide to prepare a mitochondrial division inhibitor solution; C3. Add the mitochondrial division inhibitor solution from step C2 to the Zn-CeO2@P dispersion from step C1, stir and react, then centrifuge and wash to obtain the final product.

[0011] Further, in step A1, the molar ratio of the cerium-containing precursor to the zinc-containing precursor is 1:0.5-2; the cerium-containing precursor is at least one of Ce(NO3)3·6H2O, Ce(CH3COO)3·1.5H2O, and (NH4)2Ce(NO3)6, and the zinc-containing precursor is at least one of Zn(NO3)2·6H2O, ZnCl2, ZnO, and ZnSO4·7H2O.

[0012] Furthermore, in step A2, the molar ratio of 2-aminobenzimidazole, 2-methylimidazole, and sodium formate is 2-5:35-40:2-5; in step B2, the molar ratio of phenylboronic acid or its carboxyl derivative, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1-3:1-3.

[0013] Furthermore, in step B2, the carboxyl derivative of phenylboronic acid is at least one of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid.

[0014] Furthermore, in step (3), the mitochondrial division inhibitor is at least one of Mdivi-1, lutein, mitoxantrone, and idebenone.

[0015] This invention provides the application of the above-mentioned zinc-doped cerium oxide drug-loaded nanozyme platform in the preparation of ocular surface drug delivery formulations, instruments, or devices for the treatment of dry eye disease.

[0016] The present invention also provides an ocular surface drug delivery formulation for the treatment of dry eye disease, comprising the above-mentioned zinc-doped cerium oxide drug-loaded nanozyme platform.

[0017] Furthermore, the ocular surface drug delivery preparation is an eye drop, an ocular gel, or an ocular suspension.

[0018] The present invention has the following beneficial effects: (1) At the material construction level, the invention employs a DMF / water dual-solvent system and a 2-aminobenzimidazole / 2-methylimidazole binary coordination competition system to prepare zinc-doped cerium oxide nanozymes. This differs from conventional hydrothermal methods, co-precipitation methods, sol-gel methods, impregnation methods, and high-temperature solid-phase methods. The invention achieves in-situ doping of zinc ions during the cerium oxide formation process at a lower temperature and in a shorter time. This not only facilitates the regulation of zinc doping and oxygen vacancy formation, improving the multi-enzyme antioxidant activity of cerium oxide nanozymes, but also allows for the retention or construction of nitrogen-containing functional sites on the particle surface, providing a structural basis for subsequent phenylboronic acid coupling and mitochondrial fission inhibitor loading.

[0019] (2) At the ocular surface residence level, the present invention further introduces phenylboronic acid groups on the surface of zinc-doped cerium oxide nanoparticles. Phenylboronic acid can form reversible dynamic covalent bonds with the vicinal diol structure in the corneal epithelial glycocalyx and tear film mucin, enabling the nanozyme platform to form a stable but dynamically renewable bioadhesion interface on the ocular surface, thereby prolonging the pre-corneal residence time, increasing the local drug concentration, and reducing the underutilization of drugs caused by tear clearance.

[0020] (3) At the level of mitochondrial protection and pathological cascade blockade, this invention loads a Drp1-mediated mitochondrial division inhibitor onto zinc-doped cerium oxide nanoparticles modified with phenylboronic acid. After ocular surface adhesion, cellular uptake, and drug release, the mitochondrial division inhibitor can inhibit Drp1-mediated pathological excessive mitochondrial division, improve mitochondrial morphology and function, promote the recovery of mitochondrial quality control, and thus reduce mitochondrial DNA leakage. Since mtDNA leakage is an important upstream signal for the activation of the NLRP3 inflammasome and the AIM2 inflammasome, the platform of this invention can further reduce inflammasome initiation and Caspase-1 / GSDMD-mediated pyroptosis.

[0021] (4) At the level of cellular oxidative stress regulation, zinc-doped cerium oxide nanoparticles utilize Ce 3+ / Ce 4+ The redox cycle and zinc doping-induced oxygen vacancy structure exhibit superoxide dismutase mimicry, catalase mimicry and / or peroxidase mimicry activities, which can rapidly clear abnormally elevated reactive oxygen species under dry eye-related stress conditions and reduce oxidative damage to corneal epithelial cells.

[0022] (5) The platform of this invention is suitable for preparing ocular surface drug delivery formulations and can be used for multi-target synergistic treatment of dry eye disease. This invention achieves spatiotemporal coupling regulation from oxidative stress and mitochondrial damage to inflammatory pyroptosis, and can systematically improve the complex pathological state of dry eye disease. This invention has excellent therapeutic effects and good biocompatibility in different types of dry eye animal models, and has high potential for clinical translation. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the preparation process of the zinc-doped cerium oxide nanozyme platform Zn-CeO2@PM of the present invention; Figure 2 A scanning electron microscope image of Zn-CeO2@PM prepared in Example 1; Figure 3 Transmission electron microscopy image of Zn-CeO2@PM prepared in Example 1; Figure 4 Particle size and surface potential diagram of Zn-CeO2@PM prepared in Example 1; Figure 5 The drug release curve of Zn-CeO2@PM prepared in Example 1; Figure 6 The full X-ray photoelectron spectrum and fine S2p spectrum of Zn-CeO2@PM prepared in Example 1; Figure 7 This is a comparison chart of zinc content and oxygen vacancy content between Zn-CeO2 in Example 1 and ZCDHS in the comparative example. Figure 8 The graph shows a comparison of the DPPH scavenging abilities of Zn-CeO2 in Example 1 and ZCDHS in the comparative example. Figure 9 The image shows the ocular surface retention effect evaluation of Zn-CeO2@PM in Example 1 and ZCDHS@P in the comparative example. Figure 10 This is a graph evaluating the intracellular antioxidant effect of Zn-CeO2@PM in Example 1; Figure 11 This is a graph evaluating the mitochondrial protective effect of Zn-CeO2@PM in Example 1; Figure 12 This is an evaluation diagram of the inflammasome and pyroptosis blocking effects of Zn-CeO2@PM in Example 1; Figure 13 This is a graph evaluating the therapeutic effect of Zn-CeO2@PM in a benzalkonium chloride-induced dry eye mouse model, as described in Example 1. Detailed Implementation

[0024] The examples given below are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, conditions in the examples are performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0025] The core of the material design in this invention lies in synergistically addressing two key challenges in the treatment of dry eye disease: first, after administration to the ocular surface, the drug is easily washed away by tears, blinking, and nasolacrimal duct drainage, resulting in insufficient effective exposure time to the cornea; second, the pathological process of dry eye disease involves multiple coupled processes such as oxidative stress, mitochondrial dysfunction, inflammasome activation, and corneal epithelial cell pyroptosis, making it difficult to achieve ideal efficacy with single anti-inflammatory or single antioxidant treatments. Based on this, this invention constructs a zinc-doped cerium oxide drug-loaded nanozyme platform that integrates ocular surface bioadhesion, nanozyme antioxidant activity, mitochondrial division inhibition, and pyroptosis blocking.

[0026] Example 1: Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform The preparation process is as follows: Figure 1 Specifically, it includes the following steps: 1. Preparation of zinc-doped cerium oxide nanoparticles (1) Weigh 2 mmol Ce(NO3)3·6H2O and 2 mmol Zn(NO3)2·6H2O, add them to 50 mL N,N-dimethylformamide (DMF), and stir until completely dissolved to obtain the first solution; (2) Weigh 2 mmol of 2-aminobenzimidazole, 18 mmol of 2-methylimidazole and 1.5 mmol of sodium formate, add them to 50 mL of deionized water and stir until completely dissolved to obtain the second solution; (3) Mix the first solution with the second solution and heat to 80°C under vigorous stirring for 2 hours; (4) The product obtained in step (3) is centrifuged, washed and redispersed to obtain Zn-CeO2 nanoparticles with uniform particle size.

[0027] 2. Surface modification of phenylboronic acid and loading with Mdivi-1 (1) The Zn-CeO2 nanoparticles prepared above were dispersed in pure water to obtain a Zn-CeO2 dispersion; (2) Dissolve 1 mM phenylboronic acid, 2 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 2 mM N-hydroxysuccinimide (NHS) in DMF to obtain a pre-activated mixture; (3) The pre-activated mixture was slowly added dropwise to the Zn-CeO2 dispersion and stirred for 24 hours. After the reaction was completed, the zinc-doped cerium oxide nanozyme particles modified with phenylboronic acid were obtained by centrifugation, washing and drying, and were denoted as Zn-CeO2@P. (4) Dissolve Mdivi-1 in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL Mdivi-1 solution; 3. Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform: Zinc-doped cerium oxide nanozyme particles Zn-CeO2@P were ultrasonically dispersed in purified water to prepare a Zn-CeO2@P dispersion. Mdivi-1 solution was added to the Zn-CeO2@P dispersion, and the mixture was stirred for 24 h. After the reaction was completed, the mixture was centrifuged at 11,000 rpm for 15 min, washed, and then a zinc-doped cerium oxide drug-loaded nanozyme platform was obtained, denoted as Zn-CeO2@PM.

[0028] The morphology of Zn-CeO2@PM was characterized by scanning electron microscopy; the elemental distribution of Zn-CeO2@PM was analyzed by transmission electron microscopy; the hydration particle size and surface potential of Zn-CeO2@PM were detected by dynamic light scattering; and the drug release curve of the dialysate of Zn-CeO2@PM was detected by ultraviolet-visible spectrophotometry. The results are as follows: Figure 2-5 As shown.

[0029] Depend on Figure 2 It can be seen that Zn-CeO2@PM is composed of obvious nanoparticle aggregates. Figure 3 As can be seen from the EDS element mapping, the Zn and Ce signals are uniformly distributed and spatially completely overlapped, which confirms that Zn... 2+ It has been uniformly integrated into the bulk phase, rather than enriched on the surface. The co-localization of B, O, C, N, S, and Cl signals with the particle profile provides preliminary evidence for PBA grafting and Mdivi-1 loading. High-resolution transmission electron microscopy (HRTEM) images show (see...) Figure 3 The (111) interplanar spacing of Zn-CeO2 extends to 0.317 nm, compared to 0.312 nm for standard CeO2, directly reflecting the lattice distortion caused by doping. Selected area electron diffraction (SAED) patterns show clear concentric rings with indices pointing to the (111), (200), (220), and (311) planes, confirming the preservation of the fluorite crystal system. Aberration-corrected transmission electron microscopy (AC-TEM) imaging further reveals localized lattice disorder regions within the crystal matrix, attributed to the strain field generated by zinc doping. These results demonstrate that the coordination competition system enables in-situ doping of zinc ions in cerium oxide at lower temperatures and for shorter time periods. Figure 4 It can be seen that CeO2 undergoes severe aggregation due to strong van der Waals forces and hydrogen bonding, with hydrated particles reaching a size of 1472 nm, while the sizes of Zn-CeO2 and Zn-CeO2@PM are significantly reduced (338.68 nm and 599.72 nm, respectively). Figure 5 It can be seen that Mdivi-1 loaded in Zn-CeO2@PM achieved effective drug release within 5 hours in PBS release medium, which is suitable for drug delivery to the ocular surface.

[0030] Example 2: Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform 1. Preparation of zinc-doped cerium oxide nanoparticles (1) Weigh 2 mmol Ce(CH3COO)3·1.5H2O and 1 mmol ZnCl2, add them to 50 mL N,N-dimethylformamide (DMF), and stir until completely dissolved to obtain the first solution; (2) Weigh 5 mmol of 2-aminobenzimidazole, 40 mmol of 2-methylimidazole and 5 mmol of sodium formate, add them to 50 mL of deionized water and stir until completely dissolved to obtain the second solution; (3) Mix the first solution with the second solution and heat to 60°C under vigorous stirring for 4 hours; (4) The product obtained in step (3) is centrifuged, washed and redispersed to obtain Zn-CeO2 nanoparticles with uniform particle size.

[0031] 2. Surface modification of 2-carboxyphenylboronic acid and loading of mixed drug solutions (1) The Zn-CeO2 nanoparticles prepared above were dispersed in pure water to obtain a Zn-CeO2 dispersion; (2) Dissolve 1 mM 2-carboxyphenylboronic acid, 3 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 3 mM N-hydroxysuccinimide (NHS) in DMF to obtain a pre-activated mixture; (3) The pre-activated mixture was slowly added dropwise to the Zn-CeO2 dispersion and stirred for 24 hours. After the reaction was completed, the zinc-doped cerium oxide nanozyme particles modified with phenylboronic acid were obtained by centrifugation, washing and drying, and were denoted as Zn-CeO2@P. (4) Dissolve lutein in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL lutein solution; dissolve dexamethasone in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL dexamethasone solution; dissolve human epidermal growth factor in phosphate buffer (PBS) to prepare a 500 mL solution. A mixed solution was prepared by mixing equal volumes of g / mL human epidermal growth factor solution. 3. Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform: Zinc-doped cerium oxide nanozyme particles Zn-CeO2@P were ultrasonically dispersed in purified water to prepare Zn-CeO2@P dispersion. The mixed drug solution was added to the Zn-CeO2@P dispersion and stirred for 24 h. After the reaction was completed, the mixture was centrifuged at 11,000 rpm for 15 min and washed to obtain the zinc-doped cerium oxide drug-loaded nanozyme platform, denoted as Zn-CeO2@PM.

[0032] Example 3: Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform 1. Preparation of zinc-doped cerium oxide nanoparticles (1) Weigh 2 mmol (NH4)2Ce(NO3)6 and 4 mmol ZnSO4·7H2O, add them to 50 mL N,N-dimethylformamide (DMF), and stir until completely dissolved to obtain the first solution; (2) Weigh 2 mmol of 2-aminobenzimidazole, 25 mmol of 2-methylimidazole and 3 mmol of sodium formate, add them to 50 mL of deionized water and stir until completely dissolved to obtain the second solution; (3) Mix the first solution with the second solution and heat to 100°C under vigorous stirring for 1 hour; (4) The product obtained in step (3) is centrifuged, washed and redispersed to obtain Zn-CeO2 nanoparticles with uniform particle size.

[0033] Surface modification and loading of 2,3-carboxyphenylboronic acid with mixed drug solution (1) The Zn-CeO2 nanoparticles prepared above were dispersed in pure water to obtain a Zn-CeO2 dispersion; (2) Dissolve 1 mM 3-carboxyphenylboronic acid, 3 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 3 mM N-hydroxysuccinimide (NHS) in DMF to obtain a pre-activated mixture; (3) The pre-activated mixture was slowly added dropwise to the Zn-CeO2 dispersion and stirred for 24 hours. After the reaction was completed, the zinc-doped cerium oxide nanozyme particles modified with phenylboronic acid were obtained by centrifugation, washing and drying, and were denoted as Zn-CeO2@P. (4) Dissolve mitoxantrone in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL mitoxantrone solution; dissolve human fibroblast growth factor in phosphate-buffered saline (PBS) to prepare a 500 mL solution. g / mL human fibroblast growth factor solution; dissolve sodium hyaluronate in phosphate (PBS) buffer to prepare a 10 mg / mL sodium hyaluronate solution, thus obtaining the mixed drug solution. 3. Preparation of a zinc-doped cerium oxide drug-loaded nanozyme platform: Zinc-doped cerium oxide nanozyme particles Zn-CeO2@P were ultrasonically dispersed in purified water to prepare Zn-CeO2@P dispersion. The mixed drug solution was added to the Zn-CeO2@P dispersion and stirred for 24 h. After the reaction was completed, the mixture was centrifuged at 11,000 rpm for 15 min and washed to obtain the zinc-doped cerium oxide drug-loaded nanozyme platform, denoted as Zn-CeO2@PM.

[0034] Comparative example: To further illustrate the advantages of the preparation method and the obtained material of this invention in terms of surface functionalization ability, oxygen vacancy level, antioxidant performance, and ocular surface retention ability, a zinc-doped cerium oxide hollow mesoporous sphere material reported in the prior art (Hollow-structured Zn-doped CeO2 mesoporous spheres boost enhanced antioxidant activity and synergistic bactericidal effect. DOI:10.1016 / j.colsurfb.2024.114381) was selected as a comparative example. The specific preparation process is as follows: (1) Preparation of ZCDHS material: Weigh 4.56 mmol of cerium nitrate hexahydrate and 0.046 mmol of anhydrous zinc chloride, add 2 mL of deionized water, mix and dissolve, then add 2 g of acetic acid, and sonicate for 10 min to obtain a precursor mixture. Add the precursor mixture to 60 mL of ethylene glycol, stir at room temperature until homogeneous, then transfer to a polytetrafluoroethylene-lined high-pressure reactor and react at 180 °C for 200 min. After the reaction, allow to cool naturally to room temperature, centrifuge the obtained product at 10,000 rpm for 15 min, wash 5 times each with deionized water and ethanol, and then dry overnight at 50 °C to obtain zinc-doped cerium oxide hollow mesoporous sphere material, denoted as ZCDHS.

[0035] (2) Benzylboronic acid surface modification treatment of ZCDHS: ZCDHS was treated under the same phenylboronic acid modification conditions as in Example 1. The specific steps are as follows: The obtained ZCDHS was dispersed in water to obtain a ZCDHS dispersion. 1 mM phenylboronic acid, 2 mM MEDC and 2 mM NHS were dissolved in DMF to prepare a pre-activated mixture. The pre-activated mixture was slowly added dropwise to the ZCDHS dispersion, and the mixture was stirred and reacted at room temperature for 24 h. After the reaction was completed, the material was centrifuged, washed and dried to obtain the comparative material modified with phenylboronic acid, denoted as ZCDHS@P.

[0036] Experimental Example 1: Material Characterization (1) Surface functionalization capability test X-ray photoelectron spectroscopy (XPS) was performed on Zn-CeO2@P from Example 1 and ZCDHS@P from the comparative example, focusing on the analysis of B1s, N1s, Ce3d, Zn2p and O1s signals.

[0037] Depend on Figure 6It can be seen that a distinct B1s characteristic peak was detected in the XPS spectrum of Zn-CeO2@P, indicating that phenylboronic acid was successfully grafted onto the material surface. Simultaneously, an N1s signal related to nitrogen-containing sites on the surface was observed, indicating that the preparation system of this invention can provide active sites for subsequent phenylboronic acid coupling. In contrast, no distinct B1s characteristic peak was detected in the XPS spectrum of ZCDHS@P, indicating that when using the same phenylboronic acid modification process as in this invention, it is difficult to achieve effective grafting of phenylboronic acid onto the ZCDHS surface. These results demonstrate that the ZCDHS material prepared by the hydrothermal method in the literature lacks sufficient reactive active sites on its surface, while the Zn-CeO2 material obtained in this invention through a DMF / water dual-solvent system and coordination competition regulation of 2-aminobenzimidazole / 2-methylimidazole has a more suitable structural basis for subsequent surface functionalization.

[0038] (2) Detection of zinc content and oxygen vacancy content To evaluate the differences in zinc doping degree and defect structure between Zn-CeO2 of Example 1 and ZCDHS of the comparative example, elemental content and oxygen vacancy correlation characterization analyses were performed on Zn-CeO2 and ZCDHS, respectively. Elemental content was obtained quantitatively by inductively coupled plasma atomic emission spectrometry (ICP-OES); oxygen vacancy content was obtained by electron paramagnetic spectroscopy analysis.

[0039] Depend on Figure 7 It can be seen that the zinc content in the Zn-CeO2 material obtained in Example 1 of the present invention is higher than that in the literature material ZCDHS; at the same time, its resonance absorption signal is stronger, indicating that the material of the present invention has a higher oxygen vacancy content and a richer surface defect structure.

[0040] In summary, the dual-solvent coordination competition system adopted in this invention is more conducive to the simultaneous introduction of zinc element in the formation of cerium oxide, and is conducive to obtaining nanozyme materials with higher oxygen vacancy levels, thereby providing a structural basis for enhancing their multi-enzyme mimicry activity and antioxidant properties.

[0041] Experimental Example 2: DPPH Free Radical Scavenging Ability To compare the antioxidant properties of Zn-CeO2 in Example 1 and ZCDHS in the comparative example, a DPPH free radical scavenging experiment was conducted. Specifically, Zn-CeO2 and ZCDHS dispersions of equal mass concentrations were prepared and added to an equal volume of DPPH ethanol solution. After reacting for a certain period under light-protected conditions, the absorbance change at 517 nm was measured, and the DPPH scavenging rate was calculated using the following formula: DPPH clearance rate (%) = [1 - (Sample A - Blank A) / Control A] × 100% Wherein, Sample A is the absorbance after the addition of the material and DPPH, Blank A is the absorbance of the material dispersion alone, and Control A is the absorbance of the DPPH solution alone.

[0042] Depend on Figure 8 It can be seen that, under the same experimental conditions, the Zn-CeO2 of Example 1 of this invention exhibits a significantly higher DPPH radical scavenging rate than ZCDHS, indicating that the Zn-CeO2 of this invention has superior free radical scavenging ability and antioxidant properties. Combined with the analysis results of zinc content and oxygen vacancy content, it can be seen that the higher zinc doping level and richer oxygen vacancy structure of the Zn-CeO2 of this invention contribute to enhancing its electron transfer ability and reactive oxygen species scavenging ability.

[0043] Experimental Example 3: Ocular Surface Retention Capacity To evaluate the difference in retention capacity of Zn-CeO2@P from Example 1 and ZCDHS@P from the comparative example on the ocular surface, an in vivo ocular surface retention experiment was conducted using a small animal in vivo fluorescence imaging system. Details are as follows: Zn-CeO2@P from Example 1 and ZCDHS@P from the comparative example were fluorescently labeled with Rhodamine B and prepared as dispersions of equal concentration. Healthy mice were anesthetized, and 20 μL of the fluorescently labeled material dispersion of equal concentration was dropped onto the corneal surface of each mouse. Ocular surface fluorescence images were acquired at 0, 0.5, 5, 10, 15, 20, 25, and 30 min after drug administration using an IVIS Lumina III in vivo imaging system with an excitation wavelength of 535 nm and an emission wavelength of 600 nm. The fluorescence intensity in the corneal region was then quantitatively analyzed.

[0044] Depend on Figure 9 It can be seen that the Zn-CeO2@P of the present invention maintained a high ocular surface fluorescence intensity at multiple time points after drug administration, while the fluorescence signal of ZCDHS@P rapidly decayed under tear flushing and blinking, indicating that its ocular surface residence time was short and its retention capacity was weak. These results indicate that the Zn-CeO2@P of the present invention, after modification with phenylboronic acid, can form dynamic covalent adhesion with the vicinal diol structure in the ocular surface glycocalyx and / or tear film mucin, thereby establishing a stable bioadhesion interface on the ocular surface; while the comparative ZCDHS@P, due to the difficulty in achieving effective phenylboronic acid grafting, does not possess a long-lasting ocular surface residence capacity comparable to that of the Zn-CeO2@P of the present invention.

[0045] The results of Experiments 1-3 show that, compared with ZCDHS@P, the Zn-CeO2@P of the present invention has the following advantages: ① The Zn-CeO2@P surface prepared by this invention has active sites that are more suitable for subsequent phenylboronic acid grafting, which can achieve stable surface functionalization; ②The Zn-CeO2@P of the present invention has a higher zinc doping level and oxygen vacancy content; ③ The Zn-CeO2@P of this invention has superior free radical scavenging ability and nanozyme antioxidant activity; ④ The Zn-CeO2@P of the present invention, after being modified with phenylboronic acid, exhibits significantly enhanced ocular surface bioadhesion and pre-corneal retention ability; ⑤ The Zn-CeO2@P of this invention is more suitable as a bioadhesive drug-loaded nanozyme platform for the treatment of dry eye disease.

[0046] Experiment Example 4: In vitro cellular antioxidant and cell protection experiments To investigate the ability of Zn-CeO2@PM to scavenge intracellular reactive oxygen species, acetyl 5-(and-6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, DCFH, Invitrogen, USA) was used to stain HCECs stimulated by oxidative stress (H2O2). 5 × 10⁻⁶ 3 Cells were seeded and cultured overnight, then treated with culture medium, CeO2, or Zn-CeO2@PM (10, 25, and 50 μg / mL) for 24 h. Subsequently, the supernatant in each well was replaced with 400 μM H2O2 and incubated for 2 h to simulate an intracellular oxidative stress model. Afterward, cells were placed in serum-free DMEM / F12 medium containing fluorescent probes for 30 min, followed by washing twice with PBS. Fluorescence images of HCECs were acquired using a fluorescence microscope. The average ROS fluorescence intensity of each image was semi-quantitatively determined using ImageJ software.

[0047] To further determine the ability of Zn-CeO2@PM to scavenge mitochondrial-specific reactive oxygen species (mtROS), the levels of mtROS in HCECs treated with different methods were investigated. HCECs were seeded in DMEM / F12 complete medium and cultured for 24 h. Subsequently, cells were pretreated with CeO2 or Zn-CeO2@PM (10, 25, and 50 μg / mL) for 24 h, followed by co-culturing with 400 μM H2O2 for 2 h. After washing twice with PBS, cells were treated with 1 μM MitoSOX Red (Invitrogen, USA) for 30 min. HCECs were observed and photographed under a confocal laser scanning microscope. The fluorescence intensity of MitoSOX was measured using ImageJ software to quantify mtROS levels.

[0048] Depend on Figure 10The stress-positive control (PC) group exhibited strong ROS fluorescence, while Zn-CeO2@PM treatment significantly inhibited the accumulation of both systemic and mitochondrial ROS. Notably, even at low concentrations (10 μg / mL), Zn-CeO2@PM was significantly more effective than pure Zn-CeO2. This highlights its dual therapeutic mechanism: the Mdivi-1 carrier actively inhibits pathological Drp1-mediated cell division, thereby blocking ROS production at its source; while the highly active zinc-doped cerium oxide lattice removes residual ROS.

[0049] Experiment 5: Mitochondrial Protection Experiment Changes in intracellular mitochondrial membrane potential (ΔΨm) were assessed using a mitochondrial membrane potential assay kit containing JC-1 (Beyotime, China). HCECs were cultured at 8 × 10⁸ cells per well. 3 Cells were seeded at a density of [number] cells per well in 96-well plates and allowed to adhere overnight. Zn-CeO2 or Zn-CeO2@PM (10, 25, and 50 μg / mL) was then added to the wells and incubated for 24 h. Subsequently, the cells were stimulated with 400 μM H2O2 for 2 h. They were then stained with JC-1 at 37 °C for 20 min and observed using a fluorescence microscope.

[0050] Oxidative stress and hyperosmotic stress in HCECs were simulated using H2O2 and hypertonic medium, respectively, followed by immunofluorescence detection of Drp1 to elucidate the inhibitory effect of Mdivi-1 on this key mitochondrial splitting protein under these stress conditions. HCECs were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 mcg / well in 24-well plates and cultured at 37°C for 12 h. Subsequently, cells were treated with culture medium, Zn-CeO2, and Zn-CeO2@PM for 24 h. After incubation with 400 μM H2O2 or 500 mOsm hypertonic medium, cells were fixed with 4% paraformaldehyde at room temperature for 20 min. After fixation, cells were treated with 0.1% Triton X-100 for 20 min to allow the antibody to enter the intracellular target. To block non-specific binding, cells were incubated with 1% bovine serum albumin (BSA, Beotech, China) at room temperature for 1 h. After blocking, cells were incubated with primary antibody (anti-Drp1) at 4°C for 12 h. Cells were then washed three times with PBS to remove excess reagent and incubated with Cy3-labeled anti-rabbit IgG secondary antibody at 25°C for 4 h. Finally, cell nuclei were stained with DAPI for 10 min, and the stained cells were observed and imaged using a confocal microscope.

[0051] Depend on Figure 11The loss of mitochondrial membrane potential (ΔΨm), a marker of early apoptosis, was assessed by JC-1 staining. Zn-CeO2@PM effectively reversed the stress-induced fluorescence transition from red (JC-1 aggregates, healthy) to green (JC-1 monomers, depolarized), indicating superior efficacy in maintaining ΔΨm compared to Zn-CeO2 alone. Immunofluorescence staining results showed weak Drp1 fluorescence signals in the NC group under both oxidative stress and tear hyperosmolarity-driven stress conditions. In the PC group, both stress factors significantly enhanced Drp1 fluorescence, indicating that stress induced upregulation of Drp1 expression. Although Drp1 fluorescence was lower in the Zn-CeO2 group compared to the PC group, the level was still relatively high. In contrast, the Drp1 fluorescence signal in the Zn-CeO2@PM group was weak, similar to that in the NC group. These results indicate that Zn-CeO2@PM effectively inhibits the upregulation of Drp1 induced by dry eye-related stress.

[0052] Experimental Example 6: Inflammasome and Pyroptosis Blockade Experiment To assess the effect of Zn-CeO2@PM on inhibiting the activation of the NLRP3 inflammasome, HCECs were seeded in 24-well plates and incubated with culture medium and Zn-CeO2@PM for 24 h. Subsequently, cells were stimulated with 400 μM H2O2 to induce oxidative stress in HCECs. HCECs were fixed with 4% paraformaldehyde for 30 min and then permeated with 0.5% Tween-20 for 30 min at room temperature. Cells were blocked with 5% bovine serum albumin (BSA) for 1 h, and diluted primary antibodies were mixed with BSA and incubated overnight at 4°C in a shaker. The primary antibodies included anti-NLRP3, NF-κB, Caspase-1, and IL-1β. Cells were incubated with secondary antibodies of the corresponding species at room temperature for 2 h. Finally, cell nuclei were labeled with a DAPI-containing anti-fading mounting medium. Observation was performed using a confocal laser scanning microscope.

[0053] Depend on Figure 12 It was found that in the hydrogen peroxide (H2O2)-induced oxidative stress model, the PC group exhibited a strong inflammatory cascade response, characterized by upregulated expression of NLRP3 and Caspase-1, accompanied by enhanced NF-κB nuclear translocation and a surge in the downstream pro-inflammatory cytokine IL-1β. Notably, Zn-CeO2@PM treatment significantly reversed these pathological changes, demonstrating that it can block the oxidative stress-induced inflammatory storm through a full-chain regulatory mechanism encompassing upstream transcription initiation, intermediate inflammasome assembly, and downstream cytokine secretion.

[0054] Experiment 7: Treatment Experiment of Animal Model of Dry Eye Disease A severe dry eye model was established using benzalkonium chloride (BAC, Sigma, Germany). Induction was achieved by topical instillation of 0.2% BAC (10 μL, twice daily) for 7 consecutive days to induce severe corneal epithelial defects and disrupt tear film homeostasis. Corneal epithelial defects were assessed using slit-lamp microscopy (Kanghua Ruiming, China) with corneal fluorescein staining. Fluorescein staining was performed on days 21 and 7 to confirm successful establishment of the experimental dry eye model, designated as day 0. Subsequently, on days 0, 1, 2, 3, and 4, and days 0, 1, 3, 5, and 7, observations were made under cobalt blue light using a slit-lamp microscope after instillation of 5 μL of sodium fluorescein solution. To assess treatment efficacy, mice were euthanized on day 7, and the eyeball, including the entire conjunctiva, was removed for histological examination. The eyeball was gently rinsed with physiological saline and fixed overnight in 4% paraformaldehyde. After dehydration and embedding, paraffin sections of the eyeball were cut into 5 μm thick sagittal sections. Histological changes were assessed by hematoxylin-eosin (H&E) and periodic acid-Scheffler (PAS) staining and observed under a 3D slide scanner (3D Pannoramic, 3DHISTECH, Hungary). The microscopic morphological features of the anterior segment of the eyeball, including tissue integrity, structure, and cellular infiltration, were evaluated. Depend on Figure 13 It was observed that although free cyclosporine A and CeO2 reduced the defect area, significant defect staining remained. The Zn-CeO2 group showed partial recovery, but residual defects persisted. Zn-CeO2@PM treatment significantly reduced epithelial damage, indicating that this agent has excellent therapeutic efficacy in this severe pathology. Histologically, the PC group showed the loss of 4-5 layers of corneal epithelium, extensive stromal inflammatory infiltration, and almost complete disappearance of goblet cells. Zn-CeO2@PM treatment significantly reduced inflammatory infiltration, achieved complete corneal re-epithelialization, and promoted substantial recovery of the goblet cell population. Zn-CeO2@PM demonstrated rapid, significant, and safe therapeutic effects. Compared with the control group and the single-function group, it was more effective in improving corneal epithelial integrity, reducing inflammation levels, restoring tear film homeostasis, and showed good biocompatibility.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A zinc-doped cerium oxide drug-loaded nanozyme platform, characterized in that, The zinc-doped cerium oxide drug-loaded nanozyme platform uses zinc-doped cerium oxide nanozyme particles as the antioxidant core; the surface of the zinc-doped cerium oxide nanozyme particles has a phenylboronic acid modified layer as an ocular surface adhesion interface; the phenylboronic acid modified layer is loaded with mitochondrial division regulating drugs; the mitochondrial division regulating drugs include mitochondrial division inhibitors. The preparation method of the zinc-doped cerium oxide nanoenzyme particles is as follows: A1. The first solution is prepared by mixing the cerium-containing precursor, the zinc-containing precursor and N,N-dimethylformamide. A2. Add 2-aminobenzimidazole, 2-methylimidazole and sodium formate to deionized water, mix well and prepare the second solution; A3. Mix the first solution from step A1 with the second solution from step A2, and react at 60℃-100℃ for 1-4 hours under stirring. A4. The product obtained in step A3 is centrifuged, washed and redispersed to obtain zinc-doped cerium oxide nanozyme particles.

2. The zinc-doped cerium oxide drug-loaded nanozyme platform according to claim 1, characterized in that, The mitochondrial division regulating drug further includes at least one of an anti-inflammatory drug, a repair-promoting drug, and a lubricating and protective molecule; wherein the anti-inflammatory drug is at least one of dexamethasone, diclofenac sodium, cyclosporine A, and tacrolimus; the repair-promoting drug is at least one of human epidermal growth factor and human fibroblast growth factor; and the lubricating and protective molecule is at least one of sodium hyaluronate and sodium carboxymethyl cellulose.

3. The preparation method of the zinc-doped cerium oxide drug-loaded nanozyme platform according to claim 1 or 2, characterized in that, Includes the following steps: (1) Surface modification with phenylboronic acid: B1. Disperse zinc-doped cerium oxide nanozyme particles in buffer solution to prepare Zn-CeO2 dispersion; B2. Dissolve phenylboronic acid or a carboxyl derivative of phenylboronic acid, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide in N,N-dimethylformamide to prepare a pre-activated mixture. B3. The pre-activated mixture from step B2 is added dropwise to the Zn-CeO2 dispersion from step B1. After stirring and reacting, the mixture is centrifuged, washed, and dried to obtain phenylboronic acid-modified zinc-doped cerium oxide nanoenzyme particles Zn-CeO2@P. (2) Preparation of zinc-doped cerium oxide drug-loaded nanozyme platform: C1. Zn-CeO2@P from step (1) is ultrasonically dispersed in purified water to obtain Zn-CeO2@P dispersion; C2. Dissolve the mitochondrial division inhibitor in dimethyl sulfoxide to prepare a mitochondrial division inhibitor solution; C3. Add the mitochondrial division inhibitor solution from step C2 to the Zn-CeO2@P dispersion from step C1, stir and react, then centrifuge and wash to obtain the final product.

4. The preparation method according to claim 3, characterized in that, In step A1, the molar ratio of the cerium-containing precursor to the zinc-containing precursor is 1:0.5-2; the cerium-containing precursor is at least one of Ce(NO3)3·6H2O, Ce(CH3COO)3·1.5H2O, and (NH4)2Ce(NO3)6, and the zinc-containing precursor is at least one of Zn(NO3)2·6H2O, ZnCl2, ZnO, and ZnSO4·7H2O.

5. The preparation method according to claim 3, characterized in that, In step A2, the molar ratio of 2-aminobenzimidazole, 2-methylimidazole, and sodium formate is 2-5:35-40:2-5; in step B2, the molar ratio of phenylboronic acid or its carboxyl derivative, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1-3:1-3.

6. The preparation method according to claim 3, characterized in that, The phenylboronic acid carboxyl derivative mentioned in step B2 is at least one of 2-carboxyphenylboronic acid, 3-carboxyphenylboronic acid, and 4-carboxyphenylboronic acid.

7. The preparation method according to claim 3, characterized in that, The mitochondrial division inhibitor mentioned in step (3) is at least one of Mdivi-1, lutein, mitoxantrone, and idebenone.

8. The use of the zinc-doped cerium oxide drug-loaded nanozyme platform according to claim 1 or 2 in the preparation of ocular surface drug delivery formulations, instruments or devices for the treatment of dry eye disease.

9. An ocular surface drug delivery formulation for the treatment of dry eye disease, characterized in that, Includes the zinc-doped cerium oxide drug-loaded nanozyme platform as described in claim 1 or 2.

10. The ocular surface drug delivery formulation according to claim 9, characterized in that, The ocular surface drug delivery preparation is an eye drop, an ocular gel, or an ocular suspension.

Images