Visible light catalyzed crosslinked virus-like ruthenium oxide nanoparticles for efficient transmembrane treatment of keratoconus

Abstract

The application provides visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles for efficient transmembrane treatment of keratoconus, which are mainly prepared from cetyltrimethylammonium bromide, sodium hydroxide, tetraethyl orthosilicate, cyclohexane, ruthenium chloride, phenylalanine and pharmaceutically acceptable components or adjuvants, and have virus-like nanoscale and rough morphology. The virus-like ruthenium oxide nanoparticles can efficiently cross the corneal epithelium, enter the corneal stroma, and react with ammonium persulfate to generate free radicals, oxidize the tyrosine groups in the collagen fibers of the cornea to form a di-tyrosine bond, and cross-link the collagen fibers of the cornea. The application does not require ultraviolet light irradiation, is convenient to use, is non-invasive and reduces the discomfort of patients during treatment, and has important significance for improving the compliance and quality of life of keratoconus patients.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a visible light catalytic cross-linked virus-like oxidized ruthenium nanoparticle for efficient transmembrane treatment of keratoconus. BACKGROUND

[0002] Keratoconus is a progressive corneal disease, which causes non-inflammatory progressive thinning of the central or paracentral part of the cornea and protrudes forward in a conical shape, resulting in irregular astigmatism, myopia, and even blindness. Due to the irreversibility of keratoconus, the goal of treatment is to stop disease progression and improve vision. In terms of clinical treatment methods, corneal collagen cross-linking is a relatively mature method for treating keratoconus, which has a clinical history of 20 years, and can effectively prevent or slow down the development of keratoconus. The cross-linking surgery uses a photosensitizer, riboflavin, to cross-link the corneal stroma through the corneal epithelium with the assistance of various penetration enhancers (MI2007A002162, CN102470120A, CN102164592B, and CN115177729B, etc.), irradiation of ultraviolet light, enhancement of the strength of the lamellar structure of corneal collagen fibers, balance of internal tension of the cornea, and avoidance of continuous expansion of the cornea. However, ultraviolet light irradiation can cause certain safety risks (such as irreversible damage to the corneal epithelium, corneal edema, permanent corneal opacity, and corneal endothelial failure) and discomfort to the patient. In addition, the use of penetration enhancers increases the risk of corneal cell damage. Therefore, a cross-linking agent that can efficiently cross the corneal epithelium into the corneal stroma without the need for penetration enhancers and can cross-link without the need for ultraviolet light irradiation can not only reduce damage to the ocular cornea and avoid various postoperative complications, but also improve the safety of treatment and enhance the patient's treatment experience.

[0003] Compared with traditional riboflavin as a photosensitizer, the photocatalytic activity of ruthenium-based derivatives allows efficient cross-linking reactions under lower energy light sources, promotes single electron transfer reactions under visible light catalysis, and effectively catalyzes the oxidation of tyrosine residues to achieve cross-linking of proteins and collagen fibers. Viruses in nature have highly evolved transmembrane transport mechanisms due to their unique nanoscale and biological properties, which can effectively interact with host cell membranes and promote the transmembrane transport of substances. Therefore, the present application provides a visible light catalytic cross-linked virus-like oxidized ruthenium nanoparticle for efficient transcorneal epithelium, which is expected to improve the compliance and quality of life of patients with keratoconus.

[0004] In order to further illustrate the characteristics of the present application, the applicant has further carried out relevant searches, which are explained as follows:

[0005] WO2024054165A1, discloses a method and pharmaceutical composition for treating ocular diseases, which comprises a therapeutically effective amount of a ruthenium compound and sodium persulfate; a pharmaceutically acceptable carrier;

[0006] WO2017095240A1, mentions that ruthenium and SPS (sodium persulfate) compounds are used together as photoinitiators for photopolymerization processes. The wavelength range used is between 400-700 nm, more preferably 400-450 nm. It is claimed that the method using visible light and ruthenium-SPS mixture has less phototoxicity to cells and can be used for tissue engineering. But this prior art is mainly used to make hydrogel.

[0007] From the above explanation of prior art, those skilled in the art can know that the related drugs for treating keratoconus at present have the following characteristics:

[0008] 1. Most of the prior art is based on riboflavin, which generates singlet oxygen or free radicals by ultraviolet excitation, and forms covalent cross-linking bonds between functional groups and amino acid residues on collagen fibers;

[0009] 2. WO2024054165A1 represents a method and pharmaceutical composition for treating ocular diseases, which comprises a therapeutically effective amount of a ruthenium compound and sodium persulfate; a pharmaceutically acceptable carrier.

[0010] But these two technologies have the following defects:

[0011] (1) The penetration rate of riboflavin into the corneal epithelial stroma is very low, and the eye needs to be exposed to ultraviolet light for a long time (about 30 minutes) during treatment, which increases the risk of causing irreversible damage to the corneal epithelium and various postoperative complications such as corneal edema, permanent corneal opacity, and corneal endothelial failure;

[0012] (2) Although WO2024054165A1 is based on ruthenium compounds and sodium persulfate, the synergistic effect of the two is still small, and the technology does not solve the problem of low penetration rate into the corneal epithelium. When used, the corneal epithelial layer needs to be scraped off, which can easily cause various postoperative complications such as infection and corneal melting;

[0013] (3) Most of the existing technologies only cross-link the corneal collagen, and fail to achieve tighter corneal cross-linking treatment through a two-fold cross-linking mechanism (cross-linking glycoproteins and corneal collagen fibers in the corneal stroma);

[0014] (4) Currently, most of the prior art fails to note the technical development route for preparing virus-like nanoparticles, fails to note the use of nanoscale and biological topological structure to promote the efficiency of crossing the corneal epithelium, and fails to note the use of the cation on the surface of the virus-like cationic ruthenium-based nanoparticles to realize the high-efficiency penetration into the corneal stroma through the adsorption-mediated effect of the negative electricity on the surface of the corneal epithelial cells. SUMMARY

[0015] The present application provides a visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles, which can efficiently cross the eye corneal epithelium, cross-link the corneal collagen fibers without ultraviolet light irradiation, is convenient to use, and is painless and non-invasive in the treatment of keratocnus, and has important significance for improving the compliance and quality of life of keratocnus patients.

[0016] The virus-like ruthenium oxide nanoparticles provided by the present application are mainly prepared from the following components in mass percentage: 3-5% of cetyltrimethylammonium bromide, 4-8% of sodium hydroxide, 15-25% of tetraethyl orthosilicate, 70-90% of cyclohexane, 0.5-1% of ruthenium chloride, and 0.5-15% of phenylalanine, and the remaining components are pharmaceutically acceptable components or excipients. Those skilled in the art can understand that the mass fraction is herein.

[0017] The virus-like ruthenium oxide nanoparticles provided by the present application are mainly prepared by the following steps:

[0018] Step one, preparation of virus-like silicon dioxide nanoparticles

[0019] The virus-like ruthenium oxide nanoparticles are mainly prepared from the following components: deionized water, cetyltrimethylammonium bromide, sodium hydroxide, tetraethyl orthosilicate, and cyclohexane. The cetyltrimethylammonium bromide and the sodium hydroxide are mixed and dissolved in 50-80 mL of deionized water, and after being fully stirred at a speed of 200-500 revolutions per minute at 40-80°C, a mixed solution prepared by mixing the tetraethyl orthosilicate and the cyclohexane is added dropwise, and high-speed stirring is performed at a speed of not less than 1000 revolutions per minute for 60-90 hours, and virus-like silicon dioxide nanoparticles are collected by centrifugation.

[0020] Step two, preparation of virus-like ruthenium oxide nanoparticles

[0021] The virus-like ruthenium oxide nanoparticles are mainly prepared from the following components: the virus-like silicon dioxide nanoparticles, ruthenium chloride, phenylalanine, and deionized water. The virus-like silicon dioxide nanoparticles and the ruthenium chloride are mixed and added to 50-80 mL of deionized water, and after being fully stirred at a speed of not less than 1000 revolutions per minute, the phenylalanine is added, and stirring is continued for 3-5 hours, and virus-like silicon dioxide-ruthenium nanoparticles are collected by centrifugation.

[0022] Step three, preparation of virus-like ruthenium oxide nanoparticles

[0023] The virus-like silica-ruthenium nanoparticles, sodium hydroxide and deionized water. The sodium hydroxide is dissolved in the deionized water to obtain a sodium hydroxide solution with a pH of 8-10, and the virus-like silica-ruthenium nanoparticles are immersed in a sufficient amount of sodium hydroxide solution for 30-60 minutes of etching reaction, and centrifugal collection to obtain virus-like ruthenium oxide nanoparticles with removed silica.

[0024] The application provides a use method of visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles, and further comprises 1%-2% (as understood by those skilled in the art, here refers to mass fraction or concentration, preferably mass fraction) of ammonium persulfate. The virus-like ruthenium oxide nanoparticles are first dispersed in a proper amount of deionized water, and then ultrasonic dispersion is performed in an ultrasonic cleaning machine for 300-600 seconds, and then the ammonium persulfate is added, and after sufficient stirring until the ammonium persulfate is completely dissolved, an eye solution is prepared; the eye solution is dropped on the corneal surface of a conical cornea drop by drop, and after infiltration for about 30-50 minutes, visible light is irradiated for 5-10 minutes, and the operation is repeated for 2-5 times.

[0025] Specifically, the visible light wavelength range is 450-680 nm, and the intensity is 1-3 W / cm2.

[0026] The application also relates to the use of visible light catalytic cross-linked virus-like cationic ruthenium-based nanoparticles in the preparation of an eye preparation.

[0027] Further, the eye preparation is used for preventing or treating corneal biomechanical performance degenerative diseases.

[0028] Further, the eye solution only contains visible light catalytic cross-linked virus-like cationic ruthenium-based nanoparticles and pharmaceutically acceptable adjuvants.

[0029] Further, the eye preparation is a solution preparation, and the concentration of the visible light catalytic cross-linked virus-like cationic ruthenium-based nanoparticles is 0.8-1.5 mg / mL.

[0030] Further, the pharmaceutically acceptable adjuvant is one or more selected from the group consisting of an osmotic pressure regulator, a preservative, a cosolvent, a pH regulator; the osmotic pressure regulator is selected from the group consisting of sodium chloride and glucose; the preservative is selected from the group consisting of sodium thiomersal, benzalkonium chloride, benzalkonium bromide and chlorobutanol; the cosolvent is selected from the group consisting of Tween-80 and Tween-20; and the pH regulator is selected from the group consisting of sodium hydroxide and hydrochloric acid.

[0031] Further, the concentration of the visible light catalytic cross-linked virus-like cationic ruthenium-based nanoparticles is 0.5-1.0 mg / mL.

[0032] Further, the eye preparation is 0.3ml / branch, which is composed of the following mass fractions of components: 1%-2% of ammonium sulfate, 30%-60% of the visible light catalytic cross-linked virus-like cationic ruthenium-based nanoparticles, and the remaining components are pharmaceutically acceptable components or adjuvants.

[0033] Compared with the prior art, the present application has the following beneficial effects:

[0034] 1. The visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles disclosed by the present application can efficiently enter the corneal stroma across the corneal epithelial cell membrane without scraping the patient's corneal epithelium and using various penetration enhancers by imitating the nanoscale and rough surface of viruses in nature, thereby reducing eye complications and improving patient compliance.

[0035] 2. The visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles disclosed by the present application utilize the single electron transfer reaction of ruthenium oxide under the catalysis of ammonium persulfate and visible light to further oxidize and cross-link the tyrosine groups in the corneal collagen fibers through covalent bonds, thereby avoiding damage to the eyes caused by ultraviolet light catalysis, facilitating use, being non-invasive and reducing patient discomfort during treatment, and improving patient compliance. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 It is a transmission electron microscope image of the virus-like ruthenium oxide nanoparticles of the present application;

[0037] Figure 2 It is an X-ray photoelectron spectroscopy of the virus-like ruthenium oxide nanoparticles of the present application treating keratoconus;

[0038] Figure 3 It is a visible light catalytic cross-linking schematic diagram of the virus-like ruthenium oxide nanoparticles of the present application;

[0039] Figure 4 It is a confocal microscope image of the virus-like ruthenium oxide nanoparticles of the present application crossing the corneal epithelium;

[0040] Figure 5 It is a photo of the virus-like ruthenium oxide nanoparticles of the present application treating isolated rabbit corneas. DETAILED DESCRIPTION

[0041] The technical solutions of the present application will be further described in detail below in combination with the drawings and specific implementation cases. It should be understood that the following examples are only illustrative and explanatory of the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope of protection intended by the present application.

[0042] The technical solutions in the present application will be clearly and completely described below in combination with the embodiments in the present application.

[0043] Example 1 provides a specific preparation case of the virus-like ruthenium oxide nanoparticles:

[0044] Preparation of virus-like silica nanoparticles

[0045] Take 0.75g of cetyltrimethylammonium bromide and 1mg of sodium hydroxide respectively, mix and dissolve in 60mL of deionized water, after fully stirring at 350rpm at 50℃, drop into the mixed solution prepared by mixing 4mL of tetraethyl orthosilicate and 16mL of cyclohexane, stir at 1000rpm for 72 hours, centrifugal collection to obtain virus-like silica nanoparticles, ready for use.

[0046] Preparation of virus-like ruthenium oxide nanoparticles

[0047] Take 100mg of the virus-like silica nanoparticles prepared above and 100mg of ruthenium chloride respectively, mix and disperse in 50mL of deionized water, after fully stirring at 1500rpm, add 120mg of phenylalanine, continue to stir at 1500rpm for 3 hours, centrifugal collection to obtain virus-like silica-ruthenium nanoparticles, ready for use.

[0048] Preparation of virus-like ruthenium oxide nanoparticles

[0049] Take an appropriate amount of the sodium hydroxide solution prepared by dissolving the sodium hydroxide in the deionized water to obtain a sodium hydroxide solution with a pH of 8, immerse the virus-like silica-ruthenium nanoparticles prepared above in 10mL of the sodium hydroxide solution, etch for 30 minutes, and then centrifugal collection to obtain virus-like ruthenium oxide nanoparticles with the silica removed.

[0050] Example 2 provides another specific preparation case of the virus-like ruthenium oxide nanoparticles:

[0051] (1) Preparation of virus-like silica nanoparticles

[0052] Take 1g of cetyltrimethylammonium bromide and 1mL of 0.1M sodium hydroxide solution respectively, mix and dissolve in 80mL of deionized water, after fully stirring at 500rpm at 60℃, drop into the mixed solution prepared by mixing 5mL of tetraethyl orthosilicate and 18mL of cyclohexane, stir at 1500rpm for 80 hours, centrifugal collection to obtain virus-like silica nanoparticles, ready for use.

[0053] (2) Preparation of virus-like ruthenium oxide nanoparticles

[0054] Take 100 mg of the virus-like silica nanoparticles and 120 mg of ruthenium chloride prepared above, mix and disperse in 60 mL of deionized water, after stirring at 2000 rpm, add 140 mg of phenylalanine, continue to stir at 2000 rpm for 3 hours, centrifugal collection to obtain virus-like silica-ruthenium nanoparticles, ready for use.

[0055] (3) Preparation of virus-like ruthenium oxide nanoparticles

[0056] Take an appropriate amount of sodium hydroxide and dissolve it in deionized water to prepare a sodium hydroxide solution with a pH of 9, immerse the virus-like silica-ruthenium nanoparticles prepared above in 10 mL of sodium hydroxide solution, etch for 30 minutes, and centrifugal collection to obtain virus-like ruthenium oxide nanoparticles with silica removed.

[0057] Example 3 provides a specific use method and treatment condition of the virus-like ruthenium oxide nanoparticles for visible light catalytic cross-linking treatment of ex vivo rabbit corneas. Weigh 1 mg of virus-like ruthenium oxide nanoparticles and disperse them in 1 mL of deionized water, place them in an ultrasonic cleaner and ultrasonically disperse them for 360 seconds, then add 15 μg of ammonium persulfate and stir until the ammonium persulfate is completely dissolved to prepare an eye solution. Use a dropper to draw up the eye solution and drop it onto the ocular surface of the rabbit cornea, and after 40 minutes of infiltration, irradiate it with 2 W / cm2 of 600 nm visible light for 8 minutes, and repeat this process 3 times. As shown in Figure 3 , after the ammonium persulfate is dissolved in deionized water, it reacts with the virus-like ruthenium oxide nanoparticles, and the valence of ruthenium in the ruthenium oxide changes from 3+ to 4+, and the oxidation of ammonium persulfate produces sulfonyl radicals, which further oxidize the tyrosine groups in the collagen fibers of the cornea to form di-tyrosine bonds, cross-linking the collagen fibers of the cornea. To evaluate the ability of the virus-like ruthenium oxide nanoparticles to cross the epithelium of the ex vivo rabbit cornea, we used fluorescein isothiocyanate dye to label the virus-like ruthenium oxide nanoparticles used for the treatment of ex vivo rabbit corneas, and used a confocal microscope to observe the ability of the virus-like ruthenium oxide nanoparticles to cross the epithelium of the rabbit cornea, as shown in Figure 4 , the virus-like ruthenium oxide nanoparticles can efficiently cross the epithelium of the rabbit cornea and enter the corneal stroma; to evaluate the treatment condition of the virus-like ruthenium oxide nanoparticles on the ex vivo rabbit cornea, we immersed the ex vivo rabbit cornea treated with the virus-like ruthenium oxide nanoparticles in 20 mL of 0.1% collagenase solution, as shown in Figure 5 , the ex vivo rabbit cornea treated with the virus-like ruthenium oxide nanoparticles through visible light catalytic cross-linking treatment can resist the degradation of collagenase, and still retains 60% of the cornea after 12 hours.

[0058] Comparative Example 1 provides the treatment condition of riboflavin combined with ultraviolet light for the treatment of ex vivo rabbit corneas:

[0059] A riboflavin eye solution was prepared by dissolving 1 mg of riboflavin molecules in 1 mL of deionized water, incubating at room temperature for 30 minutes, and then using a dropper to drop 300 μL of the riboflavin eye solution onto the corneal surface of a rabbit eye every 30 minutes, and then irradiating with 365 nm ultraviolet light at a strength of 3 W / cm2for 10 minutes, repeating 3 times with 10 minutes between each time. The riboflavin molecules used for treatment of the isolated rabbit corneas were dyed and labeled with fluorescein isothiocyanate, and the ability of the riboflavin molecules to cross the corneal epithelium was observed using a confocal microscope, as shown in Fig. 1. Figure 4 As shown in Fig. 2, the virus-like ruthenium oxide nanoparticles were much more efficient than the riboflavin molecules in crossing the corneal epithelium. The isolated rabbit corneas treated with riboflavin molecules combined with ultraviolet light intervention were placed in 20 mL of 0.1% collagenase solution, as shown in Fig. 3, the isolated rabbit corneas treated with riboflavin molecules combined with ultraviolet light intervention were completely degraded after 12 hours. Figure 5

[0060] In addition, it is obvious to those skilled in the art that the various components in the present application are either mass fractions or volume fractions, and the remaining components not mentioned are pharmaceutically acceptable components or excipients, which are not described here.

[0061] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not limiting; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing examples, or make equivalent substitutions for some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.​

Claims

1. Visibly light catalyzed cross-linked virus-like ruthenium oxide nanoparticles, characterized in that, Prepared from components including the following mass percentages: Hexadecyl trimethyl ammonium bromide 3%~5%, sodium hydroxide 4%~8%, tetraethyl orthosilicate 15%~25%, cyclohexane 70%~90%, ruthenium chloride 0.5%~1%, phenylalanine 0.5%~15%, and the remaining components being pharmaceutically acceptable components or excipients; The preparation method of the visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles includes the following steps: in the step of preparing the virus-like silicon dioxide nanoparticles, the raw materials used include deionized water, hexadecyl trimethyl ammonium bromide, sodium hydroxide, tetraethyl orthosilicate and cyclohexane, the hexadecyl trimethyl ammonium bromide and the sodium hydroxide are mixed and dissolved in 50-80 mL of deionized water, after being fully stirred at a speed of 200-500 revolutions per minute at 40-80°C, a mixed solution prepared by mixing the tetraethyl orthosilicate and the cyclohexane is added drop by drop, and the stirring is performed at a speed of not less than 1000 revolutions per minute for 60-90 hours, and the virus-like silicon dioxide nanoparticles are collected by centrifugation; In the step of preparing the virus-like silicon dioxide-ruthenium nanoparticles, the raw materials used include the virus-like silicon dioxide nanoparticles, ruthenium chloride, phenylalanine and deionized water, the virus-like silicon dioxide nanoparticles and the ruthenium chloride are mixed and added into 50-80 mL of deionized water, after being stirred at a speed of not less than 1000 revolutions per minute for 20-40 minutes, the phenylalanine is added, the stirring is continuously performed for 3-5 hours, and the virus-like silicon dioxide-ruthenium nanoparticles are collected by centrifugation; In the step of preparing the virus-like ruthenium oxide nanoparticles, the raw materials used include the virus-like silicon dioxide-ruthenium nanoparticles, sodium hydroxide and deionized water; the sodium hydroxide is dissolved in the deionized water to prepare a sodium hydroxide solution with a pH of 8-10, the virus-like silicon dioxide-ruthenium nanoparticles are immersed in the sodium hydroxide solution for etching reaction for 20-30 minutes, and the virus-like ruthenium oxide nanoparticles from which the silicon dioxide is removed are collected by centrifugation.

2. A preparation method for preparing the visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles according to claim 1, characterized in that: The method includes three steps of preparing the virus-like silicon dioxide nanoparticles, preparing the virus-like silicon dioxide-ruthenium nanoparticles and preparing the virus-like ruthenium oxide nanoparticles.

3. The use of the visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles prepared by the preparation method according to claim 2 in the preparation of a drug for treating keratoconus.

4. The use of the visible light catalytic cross-linked virus-like ruthenium oxide nanoparticles according to claim 1 in the preparation of an ophthalmic preparation.

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