Organic molecular cage, organic molecular cage nanoenzyme eye drop and preparation method therefor

Abstract

An organic molecular cage, an organic molecular cage nanoenzyme eye drop and a preparation method therefor, which relate to the technical field of eye drop drugs. The method for preparing the organic molecular cage comprises the following steps: S1: mixing a solution of a carboxyl-containing aliphatic o-diamine compound and an alkali solution, and ultrasonically dissolving same to obtain a transparent solution; S2: adding the transparent solution obtained in step S1 to an aqueous TFP solution and leaving same to stand; and S3: subjecting the solution after standing in step S2 to concentration, precipitation, washing, and drying sequentially to obtain the organic molecular cage. Then, the organic molecular cage is coordinated with a soluble ferrous salt and a silver salt to obtain an organic molecular cage nanoenzyme. The preparation method is simple and convenient to operate and short in preparation time. The obtained eye drop has a broad-spectrum microbicidal effect on various bacteria and fungi, and has a good treatment effect on fungal corneal infections.

Description

Organic molecular cage, organic molecular cage nanozyme eye drops and preparation method thereof Technical Field

[0001] The present invention relates to the technical field of eye drop medicines, and in particular to an organic molecular cage and a preparation method thereof, an organic molecular cage nanozyme eye drop and a preparation method thereof. Background Art

[0002] Fungal keratitis is an infectious corneal disease with a high rate of blindness caused by pathogenic fungi such as Fusarium solani, Candida albicans, Aspergillus fumigatus, and Streptomyces. Common triggers of fungal keratitis include corneal trauma, contact lens wear, and long-term use of antibiotics and corticosteroids. This infection usually leads to corneal ulcers within a few days of onset and can cause blindness if not promptly intervened. For nearly two decades, amphotericin B, voriconazole, and natamycin have been widely used in the early clinical treatment of fungal keratitis. Due to the unique anatomical and physiological structure of the cornea, these drugs often suffer from poor permeability, low solubility, and low bioavailability. To address these challenges, researchers have developed various drug delivery systems, such as microneedle array patches and chitosan derivatives, to enhance corneal permeability and improve drug bioavailability. In addition to these carriers, advanced antifungal nanomaterials, such as quaternized chitosan / silver nanoparticles / graphene oxide / voriconazole-containing contact lenses, lysosome / gallium ion integrated nanosystems, and ethylenediaminetetraacetic acid-modified AgCu2O nanoparticles, also show promise in the treatment of fungal keratitis. These solid nanoparticles can release metal ions or bioactive molecules and exhibit significant antifungal activity. Combining the advantages of drug delivery systems and advanced nanomaterials to develop advanced antifungal nanoparticle formulations that can effectively penetrate the corneal stroma is of great significance for the treatment of fungal keratitis.

[0003] Summary of the Invention

[0004] To address the poor permeability, low solubility, and low bioavailability of existing treatments for fungal keratitis, there is an urgent need to develop new antibacterial drugs. The present invention provides a method for preparing organic molecular cage nanozyme eye drops. First, in the presence of a base, trialdehyde phloroglucinol (TFP) and a carboxyl-containing aliphatic o-diamine compound undergo a [2+3] cycloimidization reaction to prepare a hydrophilic organic molecular cage. The organic molecular cage is then coordinated with a soluble ferrous salt and a silver salt to obtain an organic molecular cage nanozyme. The preparation method of the present invention is simple to operate and requires a short preparation time. The resulting eye drops have a broad-spectrum bactericidal effect against a variety of bacteria and fungi, and have excellent therapeutic effects on fungal corneal infections.

[0005] A first aspect of the present invention provides a method for preparing an organic molecular cage, the method comprising the following steps:

[0006] S1: mixing a solution of a carboxyl-containing aliphatic o-diamine compound and an alkaline solution and dissolving them by ultrasonication to obtain a transparent solution;

[0007] S2: adding the transparent solution obtained in step S1 to the TFP aqueous solution and allowing to stand;

[0008] S3: Concentrating, precipitating, washing, and drying the solution after standing in step S2 to obtain an organic molecular cage;

[0009] The molar ratio of the TFP to the carboxyl-containing aliphatic o-diamino compound is 2:3.

[0010] A second aspect of the present invention provides an organic molecular cage, which is prepared using the above-mentioned preparation method. The organic molecular cage contains carboxyl anions and is a hydrophilic organic molecular cage. The solubility of the organic molecular cage exceeds 400 mg / mL.

[0011] The third aspect of the present invention provides a method for preparing organic molecular cage nanozyme eye drops, the preparation method comprising the following steps:

[0012] S4: dissolving the organic molecular cage in deionized water to obtain an organic molecular cage solution;

[0013] S5: adding a soluble ferrous salt and a silver salt suspension to the organic molecular cage solution, and heating the mixture to obtain a mixture;

[0014] S6: dialyze and freeze-dry the mixture obtained in step S5 in sequence to obtain an organic molecular cage nanozyme; dissolve the organic molecular cage nanozyme in water to obtain an organic molecular cage nanozyme eye drop.

[0015] The mass ratio of the organic molecular cage: the soluble ferrous salt: the silver salt is 20-35: 3-7: 5-14.

[0016] The fourth aspect of the present invention provides an organic molecular cage nanozyme eye drops, which are prepared using the above-mentioned preparation method. The antibacterial rate of the organic molecular cage nanozyme eye drops is not less than 90%.

[0017] Compared with the prior art, the present invention achieves at least one of the following beneficial effects:

[0018] (1) The organic molecular cages prepared by the present invention have advantages such as good water dispersibility, peroxidase activity, in vitro and in vivo biocompatibility, and high corneal penetrability. For example, the solubility of the organic molecular cages exceeds 400 mg / mL, laying the foundation for the subsequent preparation of organic molecular cage nanozyme eye drops.

[0019] (2) The organic molecular cage nanozyme eye drops prepared by the present invention have a simple preparation method and a short preparation time; the obtained eye drops show a broad-spectrum bactericidal effect on a variety of bacteria and fungi, and have a good therapeutic effect on fungal corneal infection.

[0020] (3) The antibacterial rate of the organic molecular cage nanozyme eye drops prepared by the present invention is not less than 90%. Preferably, the antibacterial rate of the organic molecular cage nanozyme eye drops is not less than 99%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0022] FIG1 shows an exemplary embodiment of a scanning electron microscope image of an organic molecular cage according to the present invention;

[0023] FIG2 shows a synthetic route of the organic molecular cage 1# of the present invention;

[0024] FIG3 shows a mass spectrum of organic molecular cage 1#;

[0025] FIG4 shows the NMR spectrum of organic molecular cage 1#;

[0026] Figure 5 shows a transmission electron microscope photo and element mapping diagram of the organic molecular cage nanozyme in organic molecular cage nanozyme eye drops 1#;

[0027] FIG6 shows optical photographs of the organic molecular cage 1# aqueous solution and the organic molecular cage nanozyme eye drops 1# of the present invention;

[0028] FIG7 shows an in vitro antibacterial experiment diagram;

[0029] FIG8 shows a graph of the broad-spectrum antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans;

[0030] FIG9 shows an in vivo antibacterial experiment. DETAILED DESCRIPTION

[0031] In order to more clearly illustrate the overall concept of the present invention, a detailed description is given below in an exemplary manner in conjunction with the accompanying drawings.

[0032] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein, and therefore, the scope of protection of the present invention is not limited by the specific embodiments disclosed below. In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in an appropriate manner in any one or more embodiments or examples.

[0033] In an exemplary embodiment of the present invention, the method for preparing an organic molecular cage includes the following steps:

[0034] S1: mixing a solution of a carboxyl-containing aliphatic o-diamine compound and an alkaline solution and dissolving them by ultrasonication to obtain a transparent solution;

[0035] S2: adding the transparent solution obtained in step S1 to the TFP aqueous solution and allowing the mixture to stand; optionally, adding the transparent solution obtained in step S1 to the TFP aqueous solution at a rate of 10-30 drops / min.

[0036] S3: Concentrating, precipitating, washing, and drying the solution after standing in step S2 to obtain an organic molecular cage;

[0037] The molar ratio of TFP to the carboxyl-containing aliphatic vicinal diamine compound is 2:3. This ratio can ensure that TFP and the carboxyl-containing aliphatic vicinal diamine compound form a cage molecule. Too much or too little TFP and the carboxyl-containing aliphatic vicinal diamine compound will affect the formation of the cage molecule.

[0038] Optionally, the concentration of the carboxyl group-containing aliphatic vicinal diamine compound solution is 11.0-15.0 mg / mL. Preferably, the concentration of the carboxyl group-containing aliphatic vicinal diamine compound solution is 13.0 mg / mL.

[0039] Optionally, the concentration of the alkaline solution is 14.0-18.0 mg / mL. Preferably, the concentration of the alkaline solution is 16.0 mg / mL.

[0040] Optionally, the concentration of the TFP aqueous solution is 11.0-15.0 mg / mL. Preferably, the concentration of the TFP aqueous solution is 13.0 mg / mL.

[0041] Optionally, the carbon number of the aliphatic group in the carboxyl-containing aliphatic vicinal diamine compound is 3-5; preferably, the carboxyl-containing aliphatic vicinal diamine compound is 2,3-diaminopropionic acid (DAPA for short).

[0042] Optionally, the base is one or more of sodium hydroxide, potassium hydroxide, and magnesium hydroxide.

[0043] Optionally, the precipitation in step S3 is performed by adding ethanol to obtain a suspension and centrifuging to obtain a brownish yellow precipitate; and / or the washing in step S3 is performed by washing the brownish yellow precipitate with ethanol; and / or the drying in step S3 is performed by drying at 40-60°C.

[0044] Preferably, the drying temperature in step S3 is 50°C.

[0045] Specifically, the organic molecular cage contains carboxyl anions and is a hydrophilic organic molecular cage; the solubility of the organic molecular cage exceeds 400 mg / mL.

[0046] Furthermore, the organic molecular cages prepared by the present invention are water-soluble due to the presence of carboxyl anions. This allows for better metal-organic coordination between metal ions and carboxyl groups during the subsequent preparation of organic molecular cage nanozymes, ensuring a high metal ion loading rate and improving antibacterial efficacy without reducing bioavailability due to excessive metal ion content.

[0047] In another exemplary embodiment of the present invention, a method for preparing organic molecular cage nanozyme eye drops comprises the following steps:

[0048] S4: dissolving the organic molecular cage in deionized water to obtain an organic molecular cage solution;

[0049] S5: adding a soluble ferrous salt and a silver salt suspension to the organic molecular cage solution, and heating the mixture to obtain a mixture;

[0050] S6: dialyze and freeze-dry the mixture obtained in step S5 in sequence to obtain an organic molecular cage nanozyme; dissolve the organic molecular cage nanozyme in water to obtain an organic molecular cage nanozyme eye drop.

[0051] The mass ratio of the organic molecular cage: the soluble ferrous salt: the silver salt is 20-35: 3-7: 5-14.

[0052] Preferably, the organic molecular cage nanozyme obtained in step S6 is dissolved in water and then a hydrogen peroxide solution is added to obtain an organic molecular cage nanozyme eye drop. Optionally, the mass ratio of the organic molecular cage nanozyme to hydrogen peroxide is 1000:25-30.

[0053] Optionally, the concentration of the organic molecular cage solution is 20.0-30.0 mg / mL; and / or the concentration of the soluble ferrous salt solution is 3.0-6.0 mg / mL; and / or the concentration of the silver salt solution is 6.0-10.0 mg / mL.

[0054] Optionally, the soluble ferrous salt is one or more of ferrous sulfate, ferrous chloride, and ferrous nitrate; preferably, the soluble ferrous salt is ferrous sulfate.

[0055] Optionally, the silver salt is one or more of silver chloride, silver nitrate, silver sulfate, and silver acetate; preferably, the silver salt is silver acetate.

[0056] Optionally, the heating temperature in step S5 is 55-65°C for 0.75-1.25h. Preferably, the heating temperature in step S5 is 60-63°C.

[0057] Optionally, the dialysis in step S6 is performed using a 12-14 kDa dialysis bag in a large amount of distilled water for 3-5 days to remove any remaining raw materials or by-products.

[0058] Specifically, the antibacterial rate of the organic molecular cage nanozyme eye drops is not less than 90%, and the diameter of the organic molecular cage nanozyme is 30-40nm. Preferably, the antibacterial rate of the organic molecular cage nanozyme eye drops is not less than 99%.

[0059] Optionally, the iron element loading rate in the organic molecular cage nanozyme is 1.0-3.0%, and the silver element loading rate is 15.0-25.0%.

[0060] Example 1

[0061] The preparation method of the organic molecular cage is as follows:

[0062] Organic molecular cage 1#:

[0063] S1: 13.0 mg / mL LDAPA solution and 16.0 mg / mL sodium hydroxide solution were mixed and dissolved by ultrasonication to obtain a transparent solution.

[0064] S2: The transparent solution obtained in step S1 was added to a 13.0 mg / mL TFP aqueous solution at a rate of 10-30 drops / min, with a molar ratio of TFP to DAPA of 2:3, and allowed to stand at room temperature for 7 days.

[0065] S3: The solution from step S2 was concentrated using a rotary evaporator and precipitated with a large amount of ethanol. The suspension was centrifuged at 10,000 rpm for 5 minutes. The brownish-yellow precipitate was washed with ethanol and dried at 50°C for 12 hours to obtain an organic molecular cage. Referring to Figure 1, which is a scanning electron micrograph of the organic molecular cage prepared in Example 1, the organic molecular cage exhibits a rod-like stacked morphology.

[0066] Reference Figure 2 is the synthesis route of organic molecular cage 1#; Figure 3 is the mass spectrum of organic molecular cage 1#; Figure 4 is the nuclear magnetic resonance characterization of organic molecular cage 1#.

[0067] Organic molecular cage 2#:

[0068] S1: 11.0 mg / mL 3,4-diaminobutyric acid solution and 14.0 mg / mL potassium hydroxide solution were mixed and dissolved by ultrasonication to obtain a transparent solution.

[0069] S2: The transparent solution obtained in step S1 was added to an 11.0 mg / mL TFP aqueous solution, with a molar ratio of TFP to 3,4-diaminobutyric acid of 2:3, and allowed to stand at room temperature for 7 days.

[0070] S3: The solution after standing in step S2 was concentrated using a rotary evaporator and precipitated with a large amount of ethanol. The suspension was centrifuged at 10,000 rpm for 5 minutes. The brownish-yellow precipitate was washed with ethanol and dried at 40° C. for 12 hours to obtain an organic molecular cage.

[0071] Organic molecular cage 3#:

[0072] S1: 15.0 mg / mL 2,3-diaminobutyric acid and 18.0 mg / mL sodium hydroxide solution were mixed and dissolved by ultrasonication to obtain a transparent solution.

[0073] S2: The transparent solution obtained in step S1 was added dropwise to a 15.0 mg / mL TFP aqueous solution, wherein the molar ratio of TFP to 2,3-diaminobutyric acid was 2:3, and the mixture was allowed to stand at room temperature for 7 days.

[0074] S3: The solution after standing in step S2 was concentrated using a rotary evaporator and precipitated with a large amount of ethanol. The suspension was centrifuged at 10,000 rpm for 5 minutes. The brownish-yellow precipitate was washed with ethanol and dried at 60° C. for 12 hours to obtain an organic molecular cage.

[0075] Organic molecular cage 4#:

[0076] S1: 15.0 mg / mL 2,3-diaminopropionic acid and 18.0 mg / mL magnesium hydroxide solution were mixed and dissolved by ultrasonication to obtain a transparent solution.

[0077] S2: The transparent solution obtained in step S1 was added dropwise to a 15.0 mg / mL TFP aqueous solution, wherein the molar ratio of TFP to 2,3-diaminopropionic acid was 2:3, and the mixture was allowed to stand at room temperature for 7 days.

[0078] S3: The solution after standing in step S2 was concentrated using a rotary evaporator and precipitated with a large amount of ethanol. The suspension was centrifuged at 10,000 rpm for 5 minutes. The brownish-yellow precipitate was washed with ethanol and dried at 60° C. for 12 hours to obtain an organic molecular cage.

[0079] The water solubility of the organic molecular cages 1-4# prepared in Example 1 was tested, and the solubility of the organic molecular cages was more than 400 mg / mL.

[0080] Example 2

[0081] The preparation method of organic molecular cage nanozyme eye drops is as follows:

[0082] Organic molecular cage nanozyme eye drops 1#

[0083] S4: Dissolve 240.0 mg of organic molecular cage 1# in 10.0 mL of deionized water and stir at room temperature until a uniform organic molecular cage solution with a concentration of 24 mg / mL is formed.

[0084] S5: adding 5 mg / mL ferrous sulfate and 8 mg / mL silver acetate suspension to the organic molecular cage solution, and heating at 60° C. for 1 hour to obtain a mixture.

[0085] S6: The mixture obtained in step S5 is dialyzed in a large amount of distilled water using a 12-14 kDa dialysis bag for 3 days to remove any remaining raw materials or byproducts. The solution in the dialysis bag is then freeze-dried for 72 hours to obtain the organic molecular cage nanozyme. The organic molecular cage nanozyme is dissolved in water, and then a hydrogen peroxide solution is added and sonicated at room temperature until uniform, thereby obtaining the organic molecular cage nanozyme eye drops. The mass ratio of the organic molecular cage nanozyme to hydrogen peroxide is 1000:27.

[0086] Refer to Figure 5, which shows the transmission electron microscope photograph and element mapping diagram of the organic molecular cage nanozyme in organic molecular cage nanozyme eye drops 1#. A and B in Figure 5 show that the organic molecular cage nanozyme is in a dispersed, non-aggregated state, with a particle size of about 36nm; CF figures show the element distribution of silver, nitrogen, iron and oxygen respectively, proving that silver and iron elements are loaded on the organic molecular cage, and the organic molecular cage nanozyme material is successfully synthesized.

[0087] Organic molecular cage nanozyme eye drops 2#

[0088] S4: Dissolve 200.0 mg of organic molecular cage 1# in 10.0 mL of deionized water and stir at room temperature until a uniform organic molecular cage solution with a concentration of 20 mg / mL is formed.

[0089] S5: adding 3 mg / mL ferrous nitrate and 6 mg / mL silver nitrate suspension to the organic molecular cage solution, and heating at 55° C. for 1 hour to obtain a mixture.

[0090] S6: The mixture obtained in step S5 is dialyzed in a large amount of distilled water using a 12-14 kDa dialysis bag for 3 days to remove any remaining raw materials or byproducts. The solution in the dialysis bag is then freeze-dried for 72 hours to obtain the organic molecular cage nanozyme. The organic molecular cage nanozyme is dissolved in water, and then hydrogen peroxide solution is added and sonicated at room temperature until uniform, thereby obtaining the organic molecular cage nanozyme eye drops. The mass ratio of the organic molecular cage nanozyme to hydrogen peroxide is 1000:30.

[0091] Organic molecular cage nanozyme eye drops 3#

[0092] S4: 300.0 mg of organic molecular cage 2# was dissolved in 10.0 mL of deionized water and stirred at room temperature until a uniform organic molecular cage solution with a concentration of 30 mg / mL was formed.

[0093] S5: adding 6 mg / mL ferrous chloride and 10 mg / mL silver sulfate suspension to the organic molecular cage solution, and heating at 65° C. for 1 hour to obtain a mixture.

[0094] S6: The mixture obtained in step S5 is dialyzed in a large amount of distilled water using a 12-14 kDa dialysis bag for 3 days to remove any remaining raw materials or byproducts. The solution in the dialysis bag is then freeze-dried for 72 hours to obtain the organic molecular cage nanozyme. The organic molecular cage nanozyme is dissolved in water, and then a hydrogen peroxide solution is added and sonicated at room temperature until uniform, thereby obtaining the organic molecular cage nanozyme eye drops. The mass ratio of the organic molecular cage nanozyme to hydrogen peroxide is 1000:25.

[0095] Organic molecular cage nanozyme eye drops 4#

[0096] S4: Dissolve 240.0 mg of organic molecular cage 3# in 10.0 mL of deionized water and stir at room temperature until a uniform organic molecular cage solution with a concentration of 24 mg / mL is formed.

[0097] S5: adding 5 mg / mL ferrous sulfate and 8 mg / mL silver acetate suspension to the organic molecular cage solution, and heating at 60° C. for 1 hour to obtain a mixture.

[0098] S6: The mixture obtained in step S5 is dialyzed in a large amount of distilled water using a 12-14 kDa dialysis bag for 3 days to remove any remaining raw materials or byproducts. The solution in the dialysis bag is then freeze-dried for 72 hours to obtain the organic molecular cage nanozyme. The organic molecular cage nanozyme is dissolved in water, and then a hydrogen peroxide solution is added and sonicated at room temperature until uniform, thereby obtaining the organic molecular cage nanozyme eye drops. The mass ratio of the organic molecular cage nanozyme to hydrogen peroxide is 1000:27.

[0099] Organic molecular cage nanozyme eye drops 5#

[0100] Based on the organic molecular cage nanozyme eye drops 1#, the main difference is that the hydrogen peroxide solution is not added in step S6.

[0101] Comparative Example 1

[0102] Based on the organic molecular cage nanozyme eye drops 1#, the main difference is that the eye drops of comparative example 1 only contain 800umol / L hydrogen peroxide solution.

[0103] Comparative Example 2

[0104] Comparative Example 2 is a control group, which contains only physiological saline (Saline for short).

[0105] Experimental example

[0106] The performance of the organic molecular cage nanozyme eye drops 1#-5# prepared in Example 2, Comparative Example 1 and Comparative Example 2 were tested respectively.

[0107] (1) In vitro antibacterial experiment

[0108] The organic molecular cage nanozyme eye drops 1#-5# prepared in Example 2, Comparative Example 1 and Comparative Example 2 were applied to Fusarium solani, Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans for in vitro antibacterial evaluation.

[0109] The in vitro antibacterial activity of Fusarium solani (F. solani), Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans was evaluated. Briefly, each was cultured on a PDA plate at 28°C for 72 hours. Fungal spores were then collected in sterile PBS and the density was adjusted to 5×10 7Spores / mL. The spore suspension (20.0 μL) was incubated with organic molecule cage nanozyme eye drops 1#-5#, comparative example 1, and comparative example 2 at 28°C for 30 minutes. It was then diluted, spread on a PDA plate, cultured at 28°C, the fungal load was counted, and the antibacterial rate was calculated. After multiple tests and studies, the in vitro antibacterial rate of the organic molecule cage nanozyme eye drops 1#-5# prepared by the present invention was not less than 90%. Among them, the in vitro antibacterial rate of the organic molecule cage nanozyme eye drops 1#-4# was not less than 99%; the in vitro antibacterial rate of comparative example 1 was less than 10%; and the normal saline in comparative example 2 had almost no antibacterial activity.

[0110] The spore suspension and the organic molecular cage nanozyme eye drops were mixed and incubated in DCFH-DA for 30 minutes, and then centrifuged at 5000 rpm for 10 minutes. The precipitate was rinsed with PBS to remove free DCFH-DA and suspended in PBS. The suspension was then photographed using a fluorescence microscope (EchoRevolve, San Diego CA) to detect the intracellular ROS level of the fungus. Referring to Figure 7 A, there is shown a fluorescence image of the ROS level in the cells of Comparative Example 2 Saline, Comparative Example 1800umol / L hydrogen peroxide solution, organic molecular cage nanozyme eye drops 5#, and organic molecular cage nanozyme eye drops 1#.

[0111] For live / dead staining and transmission electron microscopy analysis, the organic molecular cage nanozyme eye drops and F. solani (approximately 10 7 CFU / mL) were mixed in a 50 mL centrifuge tube and incubated at 28 ° C for 16 hours. The remaining fungal cells were then collected, stained with a live / dead (SYTO9 / PI) bacterial viability kit, and fluorescence detection was performed using an echo spin hybridization microscope. In addition, some cells were fixed with 2.5% glutaraldehyde for transmission electron microscopy analysis. Referring to Figure 7 B, there are shown fluorescence images of dead / live fungal staining of Comparative Example 2 Saline, Comparative Example 1800umol / L hydrogen peroxide solution, organic molecular cage nanozyme eye drops 5#, and organic molecular cage nanozyme eye drops 1#, respectively.

[0112] Staphylococcus epidermidis (abbreviated as S.pidermidis), Staphylococcus aureus (abbreviated as S.aureus), Pseudomonas aeruginosa (abbreviated as P.aeruginosa) and Candida albicans (abbreviated as C.albicans) were cultured in LB liquid medium at 37°C overnight to evaluate the broad-spectrum antibacterial properties. The microbial suspension was mixed with the organic molecular cage nanozyme eye drops and incubated at 37°C for 30 minutes. After dilution, it was spread on the LB agar plate, cultured at 37°C overnight, and the bacterial and fungal loads were counted. Refer to Figure 7 C.

[0113] Specifically, referring to Figure 6, Figure 6 shows optical photographs of the organic molecular cage 1# aqueous solution and the organic molecular cage nanozyme eye drops 1# of the present invention from left to right. It can be seen that the organic molecular cage and the organic molecular cage nanozyme are uniformly dispersed in the solution and have good water solubility.

[0114] Figure 7 shows from left to right the in vitro antibacterial experimental diagrams of the comparative example 2 physiological saline group (referred to as Saline), the comparative example 1800umol / L hydrogen peroxide solution (referred to as H2O2 group), the organic molecular cage nanozyme eye drops 5# (referred to as the organic molecular cage nanozyme group), and the organic molecular cage nanozyme eye drops 1# (referred to as the organic molecular cage nanozyme + hydrogen peroxide group). In Figure 7, Figure A is a fluorescent image of the intracellular ROS level. It can be seen from Figure A that the ROS levels of the physiological saline group and the hydrogen peroxide group are low, and the ROS levels of the organic molecular cage nanozyme and the organic molecular cage nanozyme + hydrogen peroxide group are significantly higher; Figure B The figure shows a fluorescent image of dead / live fungal staining. From Figure B, it can be seen that the physiological saline group and the hydrogen peroxide group have more live bacteria and fewer dead bacteria, the organic molecular cage nanozyme has fewer live bacteria and more dead fungi, and the inhibition rate of the organic molecular cage nanozyme + hydrogen peroxide group is further improved. It can be seen that the organic molecular cage nanozyme itself has antibacterial properties, and its inhibition rate can be further improved by compounding with hydrogen peroxide solution. The inhibition rate of fungi is basically dead and reaches more than 99%; Figure C is a TEM image of the fungus. From Figure C, it can be seen that the fungal morphology of the physiological saline group and the hydrogen peroxide group is intact, while the fungal integrity of the organic molecular cage nanozyme and the organic molecular cage nanozyme + hydrogen peroxide group is destroyed.

[0115] Referring to Figure 8, Figure 8 shows the broad-spectrum antibacterial activity against Staphylococcus epidermidis (abbreviated as S.pidermidis), Staphylococcus aureus (abbreviated as S.aureus), Pseudomonas aeruginosa (abbreviated as P.aeruginosa) and Candida albicans (abbreviated as C.albicans). It can be seen from Figure 8 that the fungal loads of the normal saline group and the hydrogen peroxide group are both high, while the fungal loads of the organic molecular cage nanozyme group and the organic molecular cage nanozyme + hydrogen peroxide group are both low, especially the organic molecular cage nanozyme + hydrogen peroxide group has almost no fungal load.

[0116] (2) In vivo antibacterial experiment

[0117] In vivo antibacterial experiments were carried out using phosphate buffer (0.01M), voriconazole (5.0mg / mL), and organic molecular cage nanozyme eye drops 1#.

[0118] The in vivo antifungal activity of organic molecular cage nanozymes was evaluated using 8-week-old female C57BL / 6J mice. First, the mice were anesthetized by intraperitoneal injection of 0.6% sodium phenobarbital solution (1.0 mL / kg) and divided into 3 groups. A corneal epithelial scraper was used to scrape the central corneal epithelium with a diameter of 2.5 mm. In order to make a fungal keratitis model, a circular filter paper (2.5 mm in diameter) was placed on the center of the cornea and the filter paper was soaked in 10 7 The infected corneas were then treated with a phosphate-buffered saline solution, voriconazole (5.0 mg / mL), and an organic molecular cage nanozyme plus hydrogen peroxide solution (1.0 mg / mL) six times daily for 10 days. Slit lamp images of the corneas were recorded on days 1, 3, 6, and 10 to assess the progression of the infection.

[0119] The in vivo antifungal activity of the organic cage nanozyme eye drops was further evaluated in a fungal keratitis mouse model. Figure 9 shows digital images of infected corneas treated with phosphate buffer, voriconazole, and organic cage nanozyme eye drops #1. At ten days, both the phosphate buffer and voriconazole groups showed corneal edema and inflammation, while the organic cage nanozyme + hydrogen peroxide group showed only mild inflammation, demonstrating the therapeutic potential of the organic cage nanozyme + hydrogen peroxide combination against fungal keratitis induced by Fusarium solani in mice.

[0120] The foregoing is merely an embodiment of the present invention and is not intended to limit the present invention. It will be apparent to those skilled in the art that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are intended to be included within the scope of the claims of the present invention.

Claims

1. A method for preparing an organic molecular cage, characterized in that: The preparation method comprises the following steps: S1: mixing a solution of a carboxyl group-containing aliphatic ortho-diamine compound and an alkaline solution and dissolving them by ultrasonication to obtain a transparent solution; S2: adding the transparent solution obtained in step S1 into the trialdehyde phloroglucinol aqueous solution and allowing to stand; S3: sequentially concentrating, precipitating, washing and drying the solution after standing in step S2 to obtain an organic molecular cage; The molar ratio of the trialdehyde phloroglucinol to the carboxyl-containing aliphatic vicinal diamine compound is 2:3; and the carbon number of the aliphatic in the carboxyl-containing aliphatic vicinal diamine compound is 3-5.

2. The preparation method according to claim 1, characterized in that: The solution concentration of the carboxyl-containing aliphatic ortho-diamine compound is 11.0-15.0 mg / mL; and / or the concentration of the alkaline solution is 14.0-18.0 mg / mL; And / or the concentration of the trialdehyde phloroglucinol aqueous solution is 11.0-15.0 mg / mL.

3. The preparation method according to claim 1, characterized in that: The carboxyl-containing aliphatic o-diamine compound is 2,3-diaminopropionic acid.

4. The preparation method according to claim 1, characterized in that: The precipitation in step S3 is performed by adding ethanol to obtain a suspension and centrifuging to obtain a brownish yellow precipitate; and / or the washing in step S3 is washing the brownish yellow precipitate with ethanol; And / or the drying in step S3 is performed at 40-60°C.

5. An organic molecular cage, characterized in that: The organic molecular cage is prepared by the preparation method of any one of claims 1 to 4. The organic molecular cage contains carboxyl anions and is a hydrophilic organic molecular cage. The solubility of the organic molecular cage exceeds 400 mg / mL.

6. A method for preparing organic molecular cage nanozyme eye drops, characterized in that: The preparation method comprises the following steps: S4: dissolving the organic molecular cage of claim 5 in deionized water to obtain an organic molecular cage solution; S5: adding a soluble ferrous salt and a silver salt suspension to the organic molecular cage solution, and heating the mixture to obtain a mixture; S6: dialyze and freeze-dry the mixture obtained in step S5 in sequence to obtain organic molecular cage nanozyme, dissolve the organic molecular cage nanozyme in water to obtain organic molecular cage nanozyme eye drops; wherein the mass ratio of organic molecular cage: soluble ferrous salt: silver salt is 20-35: 3-7: 5-14.

7. The preparation method according to claim 6, characterized in that: The concentration of the organic molecular cage solution is 20.0-30.0 mg / mL; and / or the concentration of the soluble ferrous salt is 3.0-6.0 mg / mL; and / or the concentration of the silver salt is 6.0-10.0 mg / mL.

8. The preparation method according to claim 6, characterized in that: The soluble ferrous salt is one or more of ferrous sulfate, ferrous chloride, and ferrous nitrate; and / or the silver salt is one or more of silver chloride, silver nitrate, silver sulfate, and silver acetate.

9. The preparation method according to claim 8, characterized in that: The soluble ferrous salt is ferrous sulfate.

10. The preparation method according to claim 8, characterized in that: The silver salt is silver acetate.

11. The preparation method according to claim 6, characterized in that: The heating temperature in step S5 is 55-65°C.

12. An organic molecular cage nanozyme eye drops, characterized in that: The organic molecular cage nanozyme eye drops are prepared by any one of the preparation methods of claims 6-11, and the in vitro antibacterial rate of the organic molecular cage nanozyme eye drops is not less than 90%.

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