Bowl-shaped Janus mesoporous silica nano motor and preparation method and application thereof

Abstract

The invention provides a bowl-shaped Janus mesoporous silica nano motor and a preparation method and application thereof, and belongs to the technical field of novel biomedical materials and eye disease treatment. The preparation method comprises the following steps: preparing ZIF-8 amp by taking a ZIF-8 nanocube as a seed and taking tetraethoxysilane as a silicon source; the preparation method comprises the following steps: preparing an mSiO2 anisotropic nano hybrid, performing amino functional modification, etching to remove a ZIF-8 core to obtain amino functional bowl-shaped mesoporous silicon dioxide, and passing through B-mSiO2amp; and asymmetrically and covalently grafting catalase on the outer surface of the Janus mesoporous silica nano-motor through amino active sites on the surface of NH2 to obtain the bowl-shaped Janus mesoporous silica nano-motor. The nano-motor prepared by the invention can realize directional autonomous movement in the presence of H2O2 at an infected part, effectively enhances the penetrating ability of a drug across a corneal barrier, goes deep into corneal stroma and a bacterial biofilm, and solves the problem of low delivery efficiency of a traditional drug.

Description

Technical Field

[0001] This invention belongs to the field of novel biomedical materials and eye disease treatment technology, and particularly relates to a bowl-shaped Janus mesoporous silica nanomotor, its preparation method and application. Background Technology

[0002] Bacterial keratitis is an inflammatory disease of the cornea caused by bacterial infection and is one of the leading causes of corneal blindness. The cornea, as the outermost transparent tissue of the eyeball, plays a vital role in light transmission and refraction, but its structural integrity is highly susceptible to microbial invasion. Once bacteria invade, they release proteases and exotoxins, damaging the corneal collagen matrix and leading to serious consequences such as stromal necrosis and perforation. Simultaneously, they activate the body's immune response, recruiting immune cells to release inflammatory mediators, further exacerbating corneal damage.

[0003] Currently, clinical treatment of bacterial keratitis mainly relies on the combined use of antibiotics and anti-inflammatory drugs. However, with the increasing prevalence of multidrug-resistant (MDR) strains, the effectiveness of traditional antibiotic treatment has been significantly reduced. Furthermore, the corneal biobarrier makes it difficult for drugs to penetrate deep into the corneal stroma, biofilms formed at the infection site hinder drug contact with bacteria, and the hypoxic microenvironment caused by infection further reduces treatment efficiency. These factors collectively constitute a major challenge in the treatment of bacterial keratitis.

[0004] Corneal collagen crosslinking (CXL) technology uses riboflavin (RF) as a photosensitizer. Upon exposure to ultraviolet A (UVA), it induces covalent crosslinking of corneal stromal collagen, simultaneously generating reactive oxygen species (ROS) to exert antibacterial effects, providing a new direction for non-antibiotic treatment of multidrug-resistant bacterial infectious keratitis. However, this technology has significant limitations: firstly, riboflavin has low bioavailability, making it difficult to penetrate biological membranes and the corneal stroma to reach deeper infection sites; secondly, ROS generation is highly dependent on oxygen, and the hypoxic environment at the infection site severely weakens the antibacterial effect and collagen crosslinking efficiency.

[0005] Nanocarrier technology offers new insights for improving ocular drug delivery. Materials such as metal-organic frameworks and mesoporous silica nanoparticles have been used for drug loading and delivery, but these carriers largely rely on passive diffusion, limiting their ability to penetrate the corneal barrier. Nanomotors, as intelligent nanomaterials capable of converting chemical energy into autonomous movement, offer the possibility of active targeted drug delivery. In bacterial keratitis, elevated hydrogen peroxide (H2O2) levels in the infection microenvironment give catalase (CAT)-driven nanomotors a unique advantage. They can utilize site-specific hydrogen peroxide for autonomous propulsion, simultaneously generating oxygen (O2) to alleviate hypoxia, thus becoming highly efficient carriers for riboflavin delivery in corneal collagen cross-linking therapy. The anisotropic distribution of enzymes is crucial for optimizing the performance of catalase-driven nanomotors. However, traditional nanocarriers typically possess uniform surface chemistry, making spatially selective functionalization difficult. While interface-based masking strategies can achieve anisotropy, they involve complex multi-step synthesis processes, are inefficient, and struggle to adapt to various nanostructures. Therefore, developing a universal and simplified preparation method to achieve in-situ site-specific masking during nanoparticle synthesis is of great significance for advancing biomedical applications based on nanomotors. Summary of the Invention

[0006] To address the problems of poor drug penetration, the impact of hypoxic environment on efficacy, and the difficulty in eliminating drug-resistant bacteria in existing treatments, this invention proposes a bowl-shaped Janus mesoporous silica nanomotor, its preparation method, and its application.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a method for preparing a bowl-shaped Janus mesoporous silica nanomotor, comprising the following steps: (1) Using ZIF-8 nanocubes with truncated rhombic dodecahedral structure as seeds, in a two-phase reaction system containing aqueous and organic phases, tetraethyl orthosilicate is used as silicon source and triethylamine is used as catalyst to grow mesoporous silica on the {100} crystal plane of the ZIF-8 nanocubes to form ZIF-8&mSiO2 anisotropic nano hybrids. (2) The ZIF-8&mSiO2 anisotropic nano-hybrid was modified with amino groups to obtain ZIF-8&mSiO2&NH2; (3) The ZIF-8 core in the ZIF-8&mSiO2&NH2 is removed by etching to obtain amino-functionalized bowl-shaped mesoporous silica (B-mSiO2&NH2). (4) Catalase is asymmetrically covalently grafted onto the outer surface of the B-mSiO2&NH2 through the amino active sites on the surface of the B-mSiO2&NH2 to form a Janus-structured B-mSiO2&CAT nanomotor, which is the bowl-shaped Janus mesoporous silica nanomotor.

[0008] Further, in step (1), the organic phase is cyclohexane, the reaction temperature is 60°C, and the reaction time is 12 hours; the ratio of ZIF-8 nanocubes to tetraethyl orthosilicate is 15 mg: 100 μL.

[0009] Furthermore, in step (1), the ZIF-8 nanocubes with truncated rhombic dodecahedral (TRD) structures used are synthesized by mixing zinc salts and imidazole ligands in a solvent and controlling the reaction temperature and time, specifically including the following steps: Zn(OAc)₂·2H₂O (3.000 g) was dissolved in 50.0 mL of deionized water. In a separate beaker, 2-methylimidazole (11.700 g) and CTAB (9.7 mg) were dissolved in 50.0 mL of deionized water. The two solutions were then mixed in a 250 mL round-bottom flask and stirred. The resulting solution was allowed to stand at room temperature for 2.5 hours to obtain truncated ZIF-8 nanocubes. These nanocubes were then washed three times with deionized water and collected by centrifugation at 12,000 rpm for 6 minutes. The final product was dispersed in 100 mL of ethanol (20 mg / mL) for storage.

[0010] Furthermore, in step (1), the preparation is carried out using a two-phase method, specifically including the following steps: To a three-necked flask containing 30 mL of deionized water, add 15 mg of ZIF-8 nanocubes, 60 mg of cetyltrimethylammonium bromide (CTAB), and 30 mg of triethanolamine (TEA). Stir the mixture at 60 °C for 1 hour until a homogeneous white solution is formed. Then, add 7.0 mL of cyclohexane containing 100 μL of tetraethyl orthosilicate (TEOS) to the system to construct an aqueous / cyclohexane biphase system. Maintain magnetic stirring at 60 °C for 12 hours. After the reaction is complete, anisotropic ZIF-8 & mSiO2 nanohybrids are obtained. After centrifugation, the nanomaterials are washed alternately with deionized water and ethanol, and finally dispersed in 5.0 mL of ethanol for later use.

[0011] Furthermore, step (2), specifically the amino-functionalization modification of the anisotropic nanohybrid, includes: The ZIF-8&mSiO2 anisotropic nano-hybrid was dispersed in ethanol to obtain a dispersion of 2 mg / mL. Then (3-aminopropyl)triethoxysilane (APTES) was added, and the mixture was heated to 80°C and reacted at a constant temperature for 24 hours to obtain the ZIF-8&mSiO2&NH2. The (3-aminopropyl)triethoxysilane was used in the ZIF-8&mSiO2 anisotropic nanohybrid at a ratio of 1 mL:200 mg. Specifically, the ZIF-8&mSiO2 anisotropic nanohybrid was dispersed in 20.0 mL of anhydrous ethanol (2.0 mg / mL) and sonicated, followed by the addition of 0.2 mL of APTES. The mixture was heated to 80 °C and maintained for 24 hours. The ZIF-8&mSiO2&amino product was collected by centrifugation, washed repeatedly with water and ethanol, and finally dispersed in ethanol for subsequent use.

[0012] Further, in step (3), the step of etching away the ZIF-8 core in the ZIF-8 & mSiO2 & NH2 specifically includes: The ZIF-8&mSiO2&NH2 was dispersed in a dilute hydrochloric acid solution (pH≈3.0) and stirred for 2 hours to obtain the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2.

[0013] Furthermore, in step (4), the asymmetric grafting of catalase is achieved using the EDC / NHS chemical coupling method, specifically including: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in phosphate-buffered saline (PBS), and catalase (CAT) was added and reacted at room temperature to obtain an activated catalase solution. Subsequently, the activated catalase (CAT) solution was added to the dispersion of amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2, and stirred overnight at room temperature in the dark to achieve asymmetric grafting of catalase.

[0014] Furthermore, the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and catalase is 30:40:1.

[0015] Furthermore, the volume ratio of the activated catalase solution to the dispersion of the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2 is (3-5):1; the dispersion of the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2 is a PBS solution of the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2.

[0016] The present invention also provides a bowl-shaped Janus mesoporous silica nanomotor, which is prepared according to the above preparation method.

[0017] The present invention also provides the application of the above-mentioned bowl-shaped Janus mesoporous silica nanomotor in the preparation of a drug for treating bacterial keratitis.

[0018] Further, the method includes the step of dispersing the bowl-shaped Janus mesoporous silica nanomotor in a riboflavin aqueous solution, loading riboflavin through mesoporous adsorption, and obtaining a bowl-shaped Janus mesoporous silica nanomotor active drug delivery system.

[0019] Furthermore, through a localized ocular delivery method, the bowl-shaped Janus mesoporous silica nanomotor active drug delivery system can autonomously move under the drive of endogenous H2O2 (hydrogen peroxide) at the infection site, enhancing corneal penetration and drug delivery. Simultaneously, it catalyzes oxygen production to improve the hypoxic environment, achieving highly efficient antibacterial, anti-inflammatory, and corneal tissue repair effects in conjunction with CXL technology under UVA irradiation. The nanomotor active drug delivery system is driven by endogenous hydrogen peroxide at the infection site to autonomously move and catalyze oxygen production, followed by UVA irradiation to activate riboflavin, generate reactive oxygen species, and induce corneal collagen cross-linking.

[0020] Compared with the prior art, the present invention has the following advantages and technical effects: (1) The bowl-shaped mesoporous silica nanomotor prepared by the present invention has a unique Janus structure and CAT asymmetric modification on the outer surface of the bowl. It can achieve directional autonomous movement in the presence of H2O2 at the infection site, effectively enhancing the ability of drugs to penetrate across the corneal barrier and penetrate deep into the corneal stroma and bacterial biofilm, thus solving the problem of low efficiency of traditional drug delivery.

[0021] (2) The bowl-shaped Janus mesoporous silica nanomotor prepared in this invention catalyzes the decomposition of H2O2 to generate oxygen during the movement process, which can effectively alleviate the hypoxic microenvironment at the infection site, provide sufficient oxygen for the production of ROS by riboflavin under UVA irradiation, significantly improve the antibacterial effect and collagen crosslinking efficiency of CXL technology, and enhance the mechanical strength and anti-enzyme degradation ability of the corneal stroma.

[0022] (3) The bowl-shaped mesoporous silica nanomotor prepared by the present invention has a high specific surface area and abundant mesoporous channels, which can realize the efficient loading and sustained release of riboflavin and prolong the drug action time. At the same time, the synergistic effect of Janus structure and bowl morphology gives the nanomotor excellent motion performance and directional controllability. Its effective diffusion coefficient and motion speed are significantly better than those of traditional spherical or uniformly modified nanocarriers.

[0023] (4) The bowl-shaped mesoporous silica nanomotors obtained by the present invention have excellent biocompatibility, extremely low toxicity to human corneal epithelial cells (HCECs), and no ocular inflammation or damage to major organs was observed in in vivo experiments. They can also effectively eliminate multidrug-resistant Staphylococcus aureus (MRSA), reduce the expression of inflammatory factors (IL-1β, TNF-α, IL-6), and promote corneal tissue repair, providing a safe and effective non-antibiotic treatment strategy for multidrug-resistant bacterial infectious keratitis. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 In Figure 1, a is the SEM image of the ZIF-8 & mSiO2 anisotropic nanohybrid prepared in Example 1; b is the TEM image of B-mSiO2 prepared in Comparative Example 1; c is the SEM image of B-mSiO2; d is the structural model of B-mSiO2; e is the HAADF-STEM image of B-mSiO2 prepared in Comparative Example 1; f is the EDS elemental mapping diagram; and g is the nitrogen adsorption-desorption isotherm and pore size distribution (scale bars for a, b, and e are all 200 nm; scale bar for c is 500 nm; scale bar for f is 100 nm). Figure 2 In the diagram, a represents the four different structural nanomotors obtained in Example 1 and Comparative Examples 2-4. Specifically, a represents the flower-shaped uniformly modified F-mSiO2@CAT in Comparative Example 2, b represents the bowl-shaped uniformly modified B-mSiO2@CAT in Comparative Example 3, c represents the disc-shaped anisotropically modified D-mSiO2&CAT in Comparative Example 4, d represents the bowl-shaped anisotropically modified B-mSiO2&CAT in Example 1, eh represents the motion trajectories of the four carriers of the four different structural nanomotors (F-mSiO2@CAT in Comparative Example 2, B-mSiO2@CAT in Comparative Example 3, D-mSiO2&CAT in Comparative Example 4, and B-mSiO2&CAT in Example 1) under different H2O2 concentrations, il represents the corresponding mean square displacement (MSD) curves, mp represents the corresponding diffusion coefficient, motion velocity, and directionality quantitative analysis results, and n represents the motion direction diagram of the bowl-shaped anisotropically modified B-mSiO2&CAT nanomotor in Example 1. Figure 3In the middle section, a shows slit-lamp images and anterior segment optical coherence tomography (AS-OCT) images of MRSA-infected mice on days 0, 1, 3, 5, and 7 after different treatment groups; b shows the bacterial colony count results of corneal tissue after different treatment groups; c shows H&E staining images of corneal tissue after different treatment groups (scale bar: 200 μm); d shows the RT-qPCR quantitative analysis results of inflammatory factors IL-1β, TNF-α, and IL-6 in corneal tissue after different treatment groups. Figure 4 The results of collagen crosslinking enzyme resistance tests on corneal samples after different treatment groups in Example 1; Figure 5 The results are biomechanical test results of corneal samples after different treatments in Example 1. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] This invention provides a method for preparing a bowl-shaped Janus mesoporous silica nanomotor, comprising the following steps: (1) Using ZIF-8 nanocubes with truncated rhombic dodecahedral structure as seeds, in a two-phase reaction system containing aqueous and organic phases, tetraethyl orthosilicate is used as silicon source and triethylamine is used as catalyst to grow mesoporous silica on the {100} crystal plane of ZIF-8 nanocubes to form ZIF-8&mSiO2 anisotropic nano hybrids. (2) The ZIF-8&mSiO2 anisotropic nano-hybrid was modified with amino groups to obtain ZIF-8&mSiO2&NH2; (3) Etching removes the ZIF-8 core in ZIF-8&mSiO2&NH2 to obtain amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2; (4) Catalase is asymmetrically covalently grafted onto the outer surface of amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2 through amino active sites, forming a Janus-structured B-mSiO2&CAT nanomotor, which is a bowl-shaped Janus mesoporous silica nanomotor.

[0031] The ZIF-8 nanocube with a truncated rhombic dodecahedral structure used in this embodiment of the invention was prepared according to the technical solution described in existing literature. The specific preparation method is as follows: Zn(OAc)₂·2H₂O (3.000 g) was dissolved in 50.0 mL of deionized water. Separately, 2-methylimidazole (11.700 g) and CTAB (9.7 mg) were dissolved in 50.0 mL of deionized water in a separate beaker. The two solutions were then mixed in a 250 mL round-bottom flask and stirred. The resulting solution was allowed to stand at room temperature for 2.5 hours to obtain truncated ZIF-8 nanocubes. These were then washed three times with deionized water and collected by centrifugation at 12000 rpm for 6 minutes. The final product was dispersed in 100 mL of ethanol (20 mg / mL) for storage. The obtained truncated rhombic dodecahedral ZIF-8 nanocubes had a {100} / {110} crystal plane ratio of 0.57 and an average diameter of 300 nm.

[0032] More specifically, the method for preparing the bowl-shaped Janus mesoporous silica nanomotor in this embodiment of the invention includes the following steps: (1) Preparation of ZIF-8 & mSiO2 anisotropic nanohybrids A two-phase method was used for preparation. 15 mg of ZIF-8 nanocubes, 60 mg of cetyltrimethylammonium bromide (CTAB), and 30 mg of triethanolamine (TEA) were added to a three-necked flask containing 30 mL of deionized water. The mixture was stirred at 60 °C for 1 hour until a homogeneous white solution was formed. Subsequently, 7.0 mL of cyclohexane containing 100 μL of tetraethyl orthosilicate (TEOS) was added to the system to construct an aqueous / cyclohexane two-phase system. The reaction was maintained at 60 °C with magnetic stirring for 12 hours. After the reaction was complete, anisotropic ZIF-8 & mSiO2 nanohybrids were obtained. After centrifugation, the nanomaterials were washed alternately with deionized water and ethanol for later use.

[0033] (2) Preparation of amino-functionalized bowl-shaped mesoporous silica (B-mSiO2&NH2) The ZIF-8 & mSiO2 anisotropic nanohybrids prepared above were dispersed in 20.0 mL of ethanol to prepare a dispersion of 2 mg / mL. The dispersion was ultrasonicated until homogeneous. 0.2 mL of (3-aminopropyl)triethoxysilane (APTES) was added to the dispersion, and the mixture was heated to 80 °C and reacted at a constant temperature for 24 hours to achieve amino functionalization modification. After the reaction was completed, the product was collected by centrifugation and washed repeatedly with deionized water and ethanol to remove unreacted APTES and impurities, yielding ZIF-8 & mSiO2 & NH2. Subsequently, it was dispersed in 10 mL of dilute hydrochloric acid solution (pH≈3.0), stirred for 2 hours, and the ZIF-8 core was selectively etched away to obtain B-mSiO2 & NH2. (3) Preparation of B-mSiO2 & CAT nanomotors Catalase (CAT) was grafted onto the surface of B-mSiO2 & NH2 using the EDC / NHS activation method to prepare B-mSiO2 & CAT nanomotors. First, carboxyl activation was performed by dissolving 60.0 mg EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 80.0 mg NHS (N-hydroxysuccinimide) in 10.0 mL phosphate-buffered saline (PBS), adding 2.0 mg CAT, and reacting at room temperature for 4 hours to activate the CAT carboxyl groups. Subsequently, the activated CAT solution was added to a PBS dispersion of B-mSiO2 & NH2 (volume ratio of (3-5):1), and stirred overnight at room temperature in the dark to allow CAT to covalently bind to the amino groups on the surface of B-mSiO2 & NH2. After the reaction, the mixture was collected by centrifugation and washed multiple times with PBS to remove unbound free CAT, yielding the B-mSiO2 & CAT nanomotors.

[0034] This invention also provides a bowl-shaped Janus mesoporous silica nanomotor, prepared according to the above-described preparation method.

[0035] This invention also provides an application of the above-mentioned bowl-shaped Janus mesoporous silica nanomotor in the preparation of a drug for treating bacterial keratitis.

[0036] In this embodiment of the invention, the active drug delivery system (B-mSiO2&CAT-RF) is obtained by dispersing bowl-shaped Janus mesoporous silica nanomotors in a riboflavin aqueous solution and loading riboflavin through mesoporous adsorption. Specifically, the process includes: dispersing 20 mg of B-mSiO2&CAT nanomotors in 10 mL of a riboflavin (RF) aqueous solution with a concentration of 2 mg / mL, stirring at room temperature in the dark for 24 hours, achieving efficient loading of RF through mesoporous adsorption, collecting the product by centrifugation after the reaction is complete, washing twice with deionized water to remove the free RF adsorbed on the surface, and obtaining B-mSiO2&CAT-RF.

[0037] In this embodiment of the invention, the bowl-shaped Janus mesoporous silica nanomotor active drug delivery system, via a local ocular administration method, can autonomously move under the drive of endogenous H2O2 (hydrogen peroxide) at the infection site, enhancing corneal penetration and drug delivery. Simultaneously, it catalyzes the generation of oxygen to improve the hypoxic environment, achieving highly efficient antibacterial, anti-inflammatory, and corneal tissue repair effects in conjunction with CXL technology under UVA irradiation. The system utilizes endogenous hydrogen peroxide at the infection site to drive the autonomous movement of the nanomotor active drug delivery system and catalyze the generation of oxygen. Subsequent UVA irradiation activates riboflavin to generate reactive oxygen species and induces corneal collagen cross-linking.

[0038] In this embodiment of the invention, room temperature refers to "25±3℃".

[0039] The technical solution of the present invention will be further illustrated by the following embodiments.

[0040] Example 1 (1) Preparation of ZIF-8 & mSiO2 anisotropic nanohybrids A two-phase method was used for preparation. 15 mg of ZIF-8 nanocubes, 60 mg of CTAB, and 30 mg of TEA were added to a three-necked flask containing 30 mL of deionized water. The mixture was stirred at 60 °C for 1 hour until a homogeneous white solution was formed. Subsequently, 7.0 mL of cyclohexane containing 100 μL of TEOS was added to the system to construct a water / cyclohexane two-phase system. The reaction was carried out at 60 °C with magnetic stirring for 12 hours. After the reaction was complete, anisotropic ZIF-8 & mSiO2 nanohybrids were obtained. After centrifugation, the nanomaterials were washed alternately with deionized water and ethanol for later use.

[0041] (2) Preparation of amino-functionalized bowl-shaped mesoporous silica (B-mSiO2&NH2) The ZIF-8 & mSiO2 anisotropic nano-hybrid prepared above was dispersed in 5.0 mL of ethanol to prepare a dispersion of 2 mg / mL. The dispersion was ultrasonicated until homogeneous. 0.2 mL of APTES was added to the dispersion, and the temperature was raised to 80 °C and reacted for 24 hours to achieve amino functionalization modification. After the reaction was completed, the product was collected by centrifugation and washed repeatedly with deionized water and ethanol to remove unreacted APTES and impurities, yielding ZIF-8 & mSiO2 & NH2. Subsequently, it was dispersed in 10 mL of dilute hydrochloric acid solution (pH≈3.0), stirred for 2 hours, and the ZIF-8 core was selectively etched away to obtain B-mSiO2 & NH2. (3) Preparation of B-mSiO2 & CAT nanomotors A method for grafting CAT onto the surface of B-mSiO2 & NH2 was used to prepare B-mSiO2 & CAT nanomotors. First, carboxyl activation was performed by dissolving 60.0 mg EDC and 80.0 mg NHS in 10.0 mL phosphate buffer, adding 2.0 mg CAT, and reacting at room temperature for 4 hours to activate the CAT carboxyl groups. Subsequently, the activated CAT solution was added to a PBS dispersion of B-mSiO2 & NH2 (volume ratio 3:1), and stirred overnight at room temperature in the dark to allow CAT to covalently bind to the amino groups on the B-mSiO2 & NH2 surface. After the reaction, the mixture was collected by centrifugation and washed repeatedly with PBS to remove unbound free CAT, yielding bowl-shaped anisotropically modified B-mSiO2 & CAT nanomotors.

[0042] (4) Preparation of B-mSiO2 & CAT-RF 20 mg of B-mSiO2 & CAT nanomotors were dispersed in 10 mL of riboflavin (RF) aqueous solution with a concentration of 2 mg / mL. The mixture was stirred at room temperature in the dark for 24 hours. RF was efficiently loaded through mesoporous adsorption. After the reaction was completed, the product was collected by centrifugation and washed twice with deionized water to remove the free RF adsorbed on the surface, thus obtaining B-mSiO2 & CAT-RF.

[0043] Comparative Example 1 (1) Preparation of ZIF-8 & mSiO2 anisotropic nanohybrids A two-phase method was used for preparation. 15 mg of ZIF-8 nanocubes, 60 mg of CTAB, and 30 mg of TEA were added to a three-necked flask containing 30 mL of deionized water. The mixture was stirred at 60 °C for 1 hour until a homogeneous white solution was formed. Subsequently, 7.0 mL of cyclohexane containing 100 μL of TEOS was added to the system to construct a water / cyclohexane two-phase system. The reaction was carried out at 60 °C with magnetic stirring for 12 hours. After the reaction was complete, anisotropic ZIF-8 & mSiO2 nanohybrids were obtained. After centrifugation, the nanomaterials were washed alternately with deionized water and ethanol for later use.

[0044] (2) Preparation of bowl-shaped mesoporous silica (B-mSiO2) The ZIF-8 & mSiO2 anisotropic nanohybrid prepared above was dispersed in 10 mL of dilute hydrochloric acid solution (pH≈3.0), stirred for 2 hours, and the ZIF-8 core was selectively etched away to obtain B-mSiO2. (3) Preparation of B-mSiO2-RF 20 mg B-mSiO2 was dispersed in 10 mL of riboflavin (RF) aqueous solution with a concentration of 2 mg / mL. The mixture was stirred at room temperature in the dark for 24 hours. RF was efficiently loaded through mesoporous adsorption. After the reaction was completed, the product was collected by centrifugation and washed twice with deionized water to remove the free RF adsorbed on the surface, thus obtaining B-mSiO2-RF.

[0045] Comparative Example 2 Same as Example 1, except that the amount of TEOS used in step (1) was changed to 140 μL to obtain F-mSiO2@CAT nanomotors with uniform flower-shaped modification.

[0046] Comparative Example 3 Same as Example 1, except that the amount of TEOS used in step (1) was changed to 120 μL, and etching was performed first, followed by amination modification and CAT grafting, to obtain a bowl-shaped uniformly modified B-mSiO2@CAT nanomotor. The specific preparation method is as follows: (1) Same as Example 1, except that 7.0 mL of cyclohexane containing 120 μL TEOS was added to the system to obtain ZIF-8&mSiO2 anisotropic nano-hybrids; (2) The ZIF-8 & mSiO2 anisotropic nano-hybrid prepared above was dispersed in 10 mL of dilute hydrochloric acid solution (pH≈3.0), stirred for 2 hours, and the ZIF-8 core was selectively etched away to obtain mSiO2. (3) The mSiO2 prepared above was dispersed in 5.0 mL of ethanol to prepare a dispersion of 2 mg / mL. The dispersion was ultrasonically treated until the system was homogeneous. 0.2 mL of APTES was added to the dispersion and the temperature was raised to 80 °C and kept at a constant temperature for 24 hours to achieve amino functionalization modification and obtain B-mSiO2@NH2. (4) Dissolve 60.0 mg EDC and 80.0 mg NHS in 10.0 mL phosphate buffer, add 2.0 mg CAT, and react at room temperature for 4 hours to complete the activation of CAT carboxyl groups. Then, add the activated CAT solution to the PBS dispersion of B-mSiO2@NH2 (volume ratio of the two is 3:1), stir overnight at room temperature in the dark, so that CAT can bind to the amino groups on the surface of B-mSiO2@NH2 through covalent bonds. After the reaction is completed, collect by centrifugation, wash with PBS several times to remove unbound free CAT, and obtain bowl-shaped uniformly modified B-mSiO2@CAT nanomotors.

[0047] Comparative Example 4 Same as Example 1, except that the amount of TEOS used in step (1) is changed to 80 μL to obtain disk-shaped anisotropic modified D-mSiO2&CAT nanomotors.

[0048] Performance testing SEM images of the ZIF-8 & mSiO2 anisotropic nanohybrids prepared in Example 1 are shown below. Figure 1 TEM images of B-mSiO2 prepared in Comparative Example 1 are shown in Figure a. Figure 1 SEM images of b-mSiO2 are shown below. Figure 1 The structural model of c, B-mSiO2 is shown in [reference needed]. Figure 1 d, combined Figure 1 As can be seen from the image, after acid treatment, the ZIF-8 cores in the ZIF-8&mSiO2 nanohybrid were selectively removed, resulting in uniform mesoporous silica B-mSiO2 with a distinct bowl-shaped morphology. The average radius of the obtained bowl-shaped mesoporous silica was approximately 105 nm, the depth was approximately 141 nm, and the wall thickness was approximately 30 nm. The HAADF-STEM image of B-mSiO2 prepared in Comparative Example 1 is shown below. Figure 1 See the EDS element mapping diagram in the middle e. Figure 1 The nitrogen adsorption-desorption isotherms and pore size distribution in the middle f are shown below. Figure 1 As can be seen from the data, B-mSiO2 possesses mesoporous channels and a distinct bowl-shaped morphology. Elemental mapping confirms that silicon, oxygen, and residual zinc are uniformly distributed within the mesoporous framework of the nanobowls. Nitrogen adsorption-desorption tests show that its specific surface area is as high as 632 m². 2 / g, pore size distribution between 2-16nm ( Figure 1In the figure, the scale bars for a, b, and e are all 200 nm; the scale bar for c is 500 nm; and the scale bar for f is 100 nm.

[0049] The hydrodynamic particle size distribution diagrams of B-mSiO2&NH2, B-mSiO2&CAT prepared in Example 1 and B-mSiO2 prepared in Comparative Example 1 are shown below. Figure 1 From the h-values, it can be seen that the hydrodynamic diameter of B-mSiO2 & CAT is approximately 200 nm, which is not significantly different from that of B-mSiO2; the zeta potential test results are shown in [reference needed]. Figure 1 As shown in Figure i, the surface potential of unmodified B-mSiO2 is -20 mV, which becomes +15 mV after amino modification (B-mSiO2 & NH2), and drops to -30 mV after coupling with catalase (B-mSiO2 & CAT). The Fourier transform infrared (FTIR) spectra are shown in [Figure i]. Figure 1 As can be seen from the middle j, they are all between 1600-1700cm -1 Characteristic absorption peaks of peptide bonds in catalase, such as C=O stretching vibrations, appeared at the location.

[0050] Schematic diagrams of the four different nanomotors obtained in Example 1 and Comparative Examples 2-4 are shown below. Figure 2 In the diagram, a represents F-mSiO2@CAT with uniform flower-like modification in Comparative Example 2, b represents B-mSiO2@CAT with uniform bowl-like modification in Comparative Example 3, c represents D-mSiO2&CAT with anisotropic modification in disc-like structure in Comparative Example 4, and d represents B-mSiO2&CAT with anisotropic modification in disc-like structure in Example 1. When the amount of TEOS used is 80 μL (Comparative Example 4), the depth of the obtained disc-shaped (shallow bowl-shaped) mesoporous silica is 93 nm and the radius is 124 nm. Increasing the TEOS volume to 120 μL (Comparative Example 3) yields a bowl-shaped structure with a greater depth (141 nm) and a slightly smaller radius (105 nm). Further increasing the TEOS amount to 140 μL (Comparative Example 2) causes six bowl-shaped units to fuse to form a flower-like structure with a stable radius, but due to the increased thickness of the silicon shell, the depth further increases to approximately 545 nm.

[0051] The motility of four nanomotors was evaluated in phosphate-buffered saline (PBS) at different hydrogen peroxide concentrations (0, 25, 50, and 100 μM). The motion trajectories of the four carriers with different H2O2 concentrations were shown in the figures for Comparative Example 2 (flower-shaped uniformly modified F-mSiO2@CAT), Comparative Example 3 (bowl-shaped uniformly modified B-mSiO2@CAT), Comparative Example 4 (disc-shaped anisotropically modified D-mSiO2&CAT), and Example 1 (bowl-shaped anisotropically modified B-mSiO2&CAT). Figure 2 The mean square displacement (MSD) curves for eh are shown below. Figure 2 The quantitative analysis results of diffusion coefficient, velocity, and directionality are shown in the table below. Figure 2 The schematic diagram of the motion direction of the bowl-shaped anisotropic modified B-mSiO2&CAT nanomotor in Example 1 is shown in Figure mp. Figure 2 As can be seen, the uniformly distributed catalase in F-mSiO2@CAT nanoparticles exhibits typical Brownian motion, with no significant changes in trajectory and mean square displacement under different hydrogen peroxide concentrations. Figure 2 (e, i). Similarly, the B-mSiO2@CAT nanomotors uniformly modified with catalase showed only a slight increase in motion with increasing hydrogen peroxide concentration, indicating that the influence of geometry is limited in the absence of anisotropic enzyme distribution. Figure 2 (f, j). The anisotropically distributed D-mSiO2 & CAT nanomotors of catalase exhibited more pronounced trajectory elongation and a moderate increase in mean square displacement (f, j). Figure 2 In contrast, the B-mSiO2&CAT nanomotor, which combines a bowl-shaped structure with anisotropic modification of catalase, exhibits a significantly prolonged motion trajectory with increasing hydrogen peroxide concentration. Figure 2 The mean square displacement curves of (h, l) show a linear time-dependent increase, indicating a significant self-diffusion propagation driven by a local product gradient. The effective diffusion coefficient increases with increasing hydrogen peroxide concentration, with B-mSiO2&CAT showing the highest value, increasing from 0.06±0.002μm at 0μM. 2 / s increased to 100μM, 2.40±0.035μm 2 / s ( Figure 2 The propulsion speed showed the same trend, increasing from 2.9 ± 0.5 μm / s to 11.9 ± 1.6 μm / s. The B-mSiO2&CAT of Example 1 was approximately 300%, 114%, and 45% higher than F-mSiO2@CAT, B-mSiO2@CAT, and D-mSiO2&CAT, respectively. Directionality, quantified by trajectory linearity, also improved with increasing hydrogen peroxide concentration, and B-mSiO2&CAT of Example 1 consistently outperformed the other groups. These results collectively indicate that the combination of the bowl-shaped morphology of B-mSiO2&CAT in Example 1 and the anisotropic distribution of the enzyme significantly enhances propulsion efficiency and directional control. Figure 2 (n).

[0052] Application Example 1 Establishment and treatment of a multidrug-resistant bacterial infectious keratitis model C57BL / 6J mice were selected, and after mechanical damage to the corneal surface, 10 μL of MRSA suspension (methicillin-resistant Staphylococcus aureus suspension) was dripped in. 7An infection model was established using CFU / mL. After 24 hours, infected mice were randomly divided into 5 groups: PBS control group (Control), B-mSiO2 group (i.e., B-mSiO2 prepared in Comparative Example 1), B-mSiO2&CAT group (i.e., B-mSiO2&CAT prepared in Example 1), B-mSiO2-RF group (i.e., B-mSiO2-RF prepared in Comparative Example 1), and B-mSiO2&CAT-RF group (i.e., B-mSiO2&CAT-RF prepared in Example 1), with 6 mice in each group. 10 μL of the corresponding preparation was applied topically to the eyes of each mouse. After 30 minutes, 365 nm UVA (30 mW / cm²) was applied. 2 The cornea was irradiated for 3 minutes to obtain corneal samples. Changes in corneal transparency and thickness were observed using a slit-lamp microscope and AS-OCT on days 0, 1, 3, 5, and 7 post-infection. On day 7, mice were sacrificed, and corneal tissue was collected for bacterial colony counting, H&E staining pathological analysis, and RT-qPCR detection of inflammatory factor expression levels. Simultaneously, the anti-degradation capacity and mechanical strength of the corneal stroma were assessed using collagenase degradation experiments and biomechanical tests.

[0053] Figure 3 Table a shows slit-lamp images and anterior segment optical coherence tomography (AS-OCT) images of MRSA-infected mice on days 0, 1, 3, 5, and 7 after different treatment groups; b shows the bacterial colony count results of corneal tissue after different treatment groups; c shows H&E staining images of corneal tissue after different treatment groups (scale bar 200 μm); d shows the RT-qPCR quantitative analysis results of IL-1β, TNF-α, and IL-6 inflammatory factors in corneal tissue after different treatment groups. It can be seen that during the 7-day treatment period, monitoring disease progression showed that in the initial stage (day 0), the corneal condition was similar in all groups, exhibiting obvious opacity and swelling, with no significant difference in clinical scores. However, by day 7, the Control, B-mSiO2, and B-mSiO2&CAT treatment groups failed to overcome MRSA infection, with no improvement in corneal opacity and even signs of perforation. The B-mSiO2-RF group showed partial recovery, while the B-mSiO2&CAT-RF group showed significant improvement, with restored corneal transparency and a structure close to healthy tissue. Figure 3 (a) To quantitatively assess antibacterial efficacy, the residual bacterial load in corneal tissue was measured using the mannitol agar assay. The B-mSiO2&CAT-RF group had the lowest colony-forming unit (CFU) count, highlighting its strong in vivo antibacterial activity. Figure 3(b) Histological evaluation of corneal sections from different groups of MRSA-infected corneas further confirmed the treatment efficacy: the phosphate-buffered saline (PBS), B-mSiO2, and B-mSiO2 & CAT groups showed extensive inflammatory cell infiltration, corneal thickening, and stromal disorder; the B-mSiO2-RF group showed moderate structural recovery; while the B-mSiO2 & CAT-RF group showed reduced inflammatory infiltration and restored tissue integrity. Figure 3 (c) Given that infection severity is generally associated with elevated levels of pro-inflammatory markers such as interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6), these cytokines were analyzed using real-time quantitative reverse transcription (RT-qPCR). Figure 3 As shown in Figure d, the expression of these mediators was significantly increased in the phosphate buffer (Control group), B-mSiO2 and B-mSiO2&CAT groups, while the levels were decreased in the B-mSiO2-RF group, with the most significant decrease in the B-mSiO2&CAT-RF treatment group.

[0054] The enzyme resistance related to corneal collagen cross-linking was assessed by immersing corneal samples from different groups in a 0.2% type II collagenase solution. The results are shown in [Table missing]. Figure 4 .like Figure 4 As shown, there were significant differences in degradation rates among the treatment groups: corneal samples treated with PBS (Control), B-mSiO2, and B-mSiO2&CAT completely dissolved within 8 hours; the resistance of the B-mSiO2-RF group was enhanced and lasted for up to 24 hours; the cornea treated with B-mSiO2&CAT-RF showed the highest enzyme resistance, and complete degradation required more than 40 hours.

[0055] Biomechanical test results of different groups of corneal samples are shown below. Figure 5 As can be seen, the Young's modulus value of the B-mSiO2&CAT-RF group was significantly increased, indicating that the corneal hardness and structural enhancement effects were more significant compared with other groups.

[0056] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a bowl-shaped Janus mesoporous silica nanomotor, characterized in that, Includes the following steps: (1) Using ZIF-8 nanocubes with truncated rhombic dodecahedral structure as seeds, in a two-phase reaction system containing aqueous and organic phases, tetraethyl orthosilicate is used as silicon source and triethylamine is used as catalyst to grow mesoporous silica on the {100} crystal plane of the ZIF-8 nanocubes to form ZIF-8&mSiO2 anisotropic nano hybrids. (2) The ZIF-8&mSiO2 anisotropic nano-hybrid was modified with amino groups to obtain ZIF-8&mSiO2&NH2; (3) The ZIF-8 core in the ZIF-8&mSiO2&NH2 is removed by etching to obtain amino-functionalized bowl-shaped mesoporous silica; (4) Catalase is asymmetrically covalently grafted onto the outer surface of the amino-functionalized bowl-shaped mesoporous silica through the amino active sites, forming a Janus-structured B-mSiO2&CAT nanomotor, which is the bowl-shaped Janus mesoporous silica nanomotor.

2. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 1, characterized in that, In step (1), the organic phase is cyclohexane, the reaction temperature is 60°C, and the reaction time is 12 hours; The ratio of ZIF-8 nanocubes to tetraethyl orthosilicate is 15 mg: 100 μL.

3. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 1, characterized in that, Step (2), specifically the amino-functionalization modification of the anisotropic nano-hybrid, includes: The ZIF-8&mSiO2 anisotropic nano-hybrid was dispersed in ethanol to obtain a dispersion of 2 mg / mL. Then (3-aminopropyl)triethoxysilane was added, and the mixture was heated to 80°C and reacted at a constant temperature for 24 hours to obtain the ZIF-8&mSiO2&NH2. The (3-aminopropyl)triethoxysilane was used in the ZIF-8&mSiO2 anisotropic nanohybrid at a ratio of 1 mL: 200 mg.

4. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 1, characterized in that, Step (3), specifically the etching process to remove the ZIF-8 core from the ZIF-8 & mSiO2 & NH2, includes: The ZIF-8&mSiO2&NH2 was dispersed in a dilute hydrochloric acid solution and stirred for 2 hours to obtain the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2.

5. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 1, characterized in that, In step (4), the asymmetric grafting of catalase is achieved using the EDC / NHS chemical coupling method, specifically including: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved in phosphate buffer, and catalase was added and reacted at room temperature to obtain an activated catalase solution. Subsequently, the activated catalase solution was added to the dispersion of amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2, and stirred overnight at room temperature in the dark to achieve asymmetric grafting of catalase.

6. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 5, characterized in that, The mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and catalase is 30:40:

1.

7. The method for preparing the bowl-shaped Janus mesoporous silica nanomotor according to claim 5, characterized in that, The volume ratio of the activated catalase solution to the dispersion of the amino-functionalized bowl-shaped mesoporous silica B-mSiO2&NH2 is (3-5):

1.

8. A bowl-shaped Janus mesoporous silica nanomotor, characterized in that, It is prepared according to any one of claims 1-7.

9. The use of the bowl-shaped Janus mesoporous silica nanomotor as described in claim 8 in the preparation of a drug for treating bacterial keratitis.

10. The application of the bowl-shaped Janus mesoporous silica nanomotor according to claim 9 in the preparation of a drug for treating bacterial keratitis, characterized in that, The method includes the steps of dispersing the bowl-shaped Janus mesoporous silica nanomotor in a riboflavin aqueous solution, loading riboflavin through mesoporous adsorption, and obtaining a bowl-shaped Janus mesoporous silica nanomotor active drug delivery system.

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