An artificial cornea applying Catalase@nHOFs nanoparticles

Abstract

The present invention discloses an artificial cornea utilizing Catalase@nHOFs nanoparticles, belonging to the field of in vivo prosthetic implant technology. This method eliminates the need for frequent dosing and provides long-lasting protection against ROS. Furthermore, it eliminates the need to adjust or alter the existing artificial cornea structure. The portion of the artificial cornea that contacts the eye is coated with a Catalase@nHOFs nanocoating. The coating is prepared by encapsulating catalase within a hydrogen-bonded organic framework to form Catalase@nHOFs nanoparticles, which adhere to the surface of the artificial cornea to form the Catalase@nHOFs nanocoating.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vivo prosthesis implantation, and in particular relates to an artificial cornea using Catalase@nHOFs nanoparticles. Background Art

[0002] Keratoprosthesis is the last and only hope for restoring vision for patients with end-stage corneal blindness. However, implantation of these implants often induces a persistent foreign body reaction, characterized by an abnormal increase in reactive oxygen species (ROS). This can lead to postoperative complications such as sterile endophthalmitis and optic nerve damage, compromising long-term efficacy. Therefore, suppressing the pathological increase in ROS after keratoprosthesis surgery is crucial for maintaining long-term efficacy.

[0003] The development of anti-ROS drugs has always been an international hot topic and a difficult issue. Currently, there are no anti-ROS drugs in ophthalmology. The drugs under development (such as hydrogen water) have poor stability, limited duration of action, and require frequent administration. In addition, after artificial cornea surgery, the ocular surface is covered with dense tissues such as ear cartilage or eyelids. The efficiency of local eye drops is extremely low, and it is impossible to use administration methods such as subconjunctival or intravitreal injections. The ocular bioavailability of systemic drugs is low and the side effects are large. Therefore, how to effectively deliver drugs into the artificial cornea eye is also a problem.

[0004] In view of this, how to effectively deliver drugs that can provide long-term anti-ROS effects into the artificial cornea to inhibit the pathological increase of ROS after surgery, improve the inflammatory state, and maintain the long-term efficacy of the artificial cornea is a technical problem that needs to be solved urgently.

[0005] Catalase, which catalyzes the decomposition of hydrogen peroxide into oxygen and water, is the most powerful tool for organisms to fight ROS. However, catalase is prone to denaturation and reduced activity in pathological environments.

[0006] A long-acting anti-ROS drug based on HOF was prepared and coated onto an artificial cornea (PMMA material) to develop an artificial cornea with long-term anti-ROS effect. Summary of the Invention

[0007] The purpose of the present invention is to provide an artificial cornea using Catalase@nHOFs nanoparticles, which does not require frequent administration and can achieve long-term anti-ROS. In addition, there is no need to adjust or change the existing artificial cornea structure.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides an artificial cornea with a Catalase@nHOFs nanocoating, wherein the portion of the artificial cornea that contacts the interior of the eye is provided with the Catalase@nHOFs nanocoating; the Catalase@nHOFs nanocoating is prepared by encapsulating catalase in a hydrogen-bonded organic framework to form Catalase@nHOFs nanoparticles, which then adhere to the surface of the artificial cornea to form the Catalase@nHOFs nanocoating.

[0010] As a possible implementation method, the thickness of the Catalase@nHOFs nanocoating is 0.5um to 50um.

[0011] As a possible implementation method, hydrogen-bonded organic frameworks encapsulate catalase to prepare Catalase@nHOFs nanoparticles, which specifically includes the following steps:

[0012] Tetramethyleneimine and azophthalate are mixed in aqueous solution and self-assembly is achieved by hydrogen bonding interaction between the two.

[0013] Catalase is prepared in a mixture of tetramethane, azophthalate and aqueous solution, and in situ encapsulation of catalase is achieved during the self-assembly process to prepare Catalase@nHOFs nanoparticles.

[0014] As a possible implementation, Catalase@nHOFs nanoparticles are in the form of nanorods with a length of 200 nm.

[0015] As a possible implementation method, the Catalase@nHOFs nanoparticles are adhered to the surface of the artificial cornea using tissue adhesive.

[0016] As a possible implementation method, the tissue adhesive includes at least biomimetic recombinant type III mussel mucin, recombinant mussel mucin rMfp151, iron-based mussel protein, mussel mucin modified by glycosylation, medical tissue adhesive based on mussel mucin and hyaluronic acid, and mussel mucin obtained by tyrosinase oxidation of mussel mucin intermediates mlp353, mlp316 and mlp319.

[0017] As a possible implementation method, the biomimetic recombinant type III mussel mucin includes a protein that constitutes the mussel byssus fiber skeleton and an Mfp-3 protein expressed in fusion with the protein. Some or all of the tyrosine residues of the Mfp-3 protein are modified into dopa groups by the action of co-expressed tyrosinase.

[0018] As a possible implementation method, the recombinant mussel mucin rMfp151 is a fusion of the Mcfp-5 mature peptide of the thick-shell mussel Mytilus coruscus and the Mefp-1 peptide of the blue mussel Mytilus edulis at both ends.

[0019] As a possible implementation manner, a hydroxyapatite layer is further plated between the tissue adhesive and the surface of the artificial cornea.

[0020] As a possible implementation, the tissue adhesive has a lap-shear tensile strength of 0.23-0.24 MPa.

[0021] Compared with the prior art, the present invention has the following effects:

[0022] 1. A Catalase@nHOFs nanocoating is applied to the portion of the artificial cornea that contacts the eye. The raw material for the Catalase@nHOFs nanocoating is Catalase@nHOFs nanoparticles, which are prepared by encapsulating catalase within a hydrogen-bonded organic framework (HOF). Encapsulating catalase within the hydrogen-bonded organic framework (HOF) provides a protective environment that effectively maintains catalase activity. Furthermore, the Catalase@nHOFs nanoparticles are efficiently taken up by cells, allowing them to stably and long-term enzymatically catalyze reactions within the cells, effectively reducing ROS. Furthermore, the Catalase@nHOFs nanoparticles can form hydrogen bonds with solid-phase materials such as polymethyl methacrylate (PMMA), stably combining to form an implantable enzyme-catalyzed anti-ROS drug. PMMA is a commonly used material for artificial corneas. Therefore, combining Catalase@nHOFs nanoparticles with PMMA and applying them to artificial corneas can effectively overcome the problems of existing artificial corneas, such as the need for frequent dosing and low drug delivery efficiency.

[0023] 2. The Catalase@nHOFs nanocoating is adhered to the artificial cornea by using mussel mucin, which has the advantages of high adhesion strength and good biocompatibility. In addition, the mussel mucin is modified, such as adding Fe 3+ It can also enhance the wear and corrosion resistance of artificial corneas. For example, by glycosylation of mussel mucin to obtain more microstructures, and then adhering Catalase@nHOFs nanoparticles to the artificial cornea, it is possible to achieve long-term sustained release of Catalase@nHOFs nanoparticles with higher adhesion strength.

[0024] 3. The present invention also coats hydroxyapatite between the mussel mucin and the artificial cornea surface (hydroxyapatite is coated on the sides of the lens column and the bracket). The irregular and diverse surface structure of hydroxyapatite makes the mussel mucin easier to adhere to, that is, it is not easily washed away or metabolized, ensuring the stability of the Catalase@nHOFs coating, thereby enabling the long-term release of catalase.

[0025] 4. Using 2',7'-dichlorodihydrofluorescein diacetate staining and imaging, as well as flow cytometry analysis, it was found that HOF-encapsulated catalase significantly reduced cellular ROS levels, significantly reducing cellular oxidative stress and increasing cell survival by 60%. Cellular experiments demonstrated that HOF-encapsulated catalase showed no tendency to degrade, suggesting long-term efficacy. DETAILED DESCRIPTION

[0026] To facilitate a clear description of the technical solutions of the embodiments of the present invention, the words "first" and "second" are used in the embodiments of the present invention to distinguish between identical or similar items with substantially the same functions and effects. For example, the first threshold and the second threshold are merely used to distinguish between different thresholds and do not limit their order. Those skilled in the art will understand that the words "first" and "second" do not limit the quantity or execution order, and the words "first" and "second" do not necessarily mean different.

[0027] It should be noted that, in the present invention, words such as "exemplary" or "for example" are used to indicate examples, illustrations, or descriptions. Any embodiment or design described as "exemplary" or "for example" in the present invention should not be construed as being preferred or advantageous over other embodiments or designs. Rather, the use of words such as "exemplary" or "for example" is intended to present the relevant concepts in a concrete manner.

[0028] In the present invention, "at least one" refers to one or more, and "more" refers to two or more. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent: the existence of A alone, the existence of A and B at the same time, and the existence of B alone, where A and B can be singular or plural. The character " / " generally indicates that the previous and next associated objects are in an "or" relationship. "At least one of the following items" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, at least one of a, b or c can represent: a, b, c, the combination of a and b, the combination of a and c, the combination of b and c, or the combination of a, b and c, where a, b, c can be single or multiple.

[0029] The embodiment of the present invention provides an artificial cornea using Catalase@nHOFs nanoparticles, which does not require frequent administration and can achieve long-term resistance to ROS. In addition, there is no need to adjust or change the existing artificial cornea structure.

[0030] The present invention provides an artificial cornea with a Catalase@nHOFs nanocoating, wherein the portion of the artificial cornea that contacts the interior of the eye is coated with the Catalase@nHOFs nanocoating; the Catalase@nHOFs nanocoating is prepared by encapsulating catalase within a hydrogen-bonded organic framework to form Catalase@nHOFs nanoparticles, which adhere to the surface of the artificial cornea to form the Catalase@nHOFs nanocoating.

[0031] A Catalase@nHOFs nanocoating is applied to the portion of the artificial cornea that contacts the eye. The raw material for the Catalase@nHOFs nanocoating is Catalase@nHOFs nanoparticles, which are prepared by encapsulating catalase within a hydrogen-bonded organic framework (HOF). Encapsulating catalase within the hydrogen-bonded organic framework (HOF) provides a protective environment that effectively maintains catalase activity. Furthermore, the Catalase@nHOFs nanoparticles are efficiently taken up by cells, allowing them to stably and long-term enzymatically catalyze reactions within the cells, effectively reducing ROS. Furthermore, the Catalase@nHOFs nanoparticles can form hydrogen bonds with solid-phase materials such as polymethyl methacrylate (PMMA), stably combining to form an implantable enzyme-catalyzed anti-ROS drug. PMMA is a commonly used material for artificial corneas. Therefore, combining Catalase@nHOFs nanoparticles with PMMA and applying them to artificial corneas can effectively overcome the problems of existing artificial corneas, such as the need for frequent dosing and low drug delivery efficiency, while achieving long-term ROS reduction.

[0032] As a possible implementation, the thickness of the Catalase@nHOFs nanocoating ranges from 0.5um to 50um, specifically 0.5um, 1.0um, 5um, 10um, 15um, 20um, 25um, 30um, 35um, 40um, 45um, or 50um. The thickness of the nanocoating can be determined based on the patient's ocular condition. For example, a thicker Catalase@nHOFs nanocoating can be selected for patients requiring prolonged drug administration, while a thinner one can be selected for patients requiring prolonged drug administration.

[0033] As a possible implementation approach, hydrogen-bonded organic frameworks (HOFs) were used to encapsulate catalase to form Catalase@nHOFs nanoparticles. The method involves the following steps: tetramethane (TAM) and azophthalate (AZB) are mixed in an aqueous solution and self-assembled using hydrogen bonding interactions between the two. Catalase is then prepared in the mixture of TAM, AZB, and aqueous solution, and in situ encapsulation of catalase occurs during the self-assembly process, resulting in the formation of Catalase@nHOFs nanoparticles. Studies have shown that hydrogen bonding interactions between protein residues and the building blocks of the hydrogen-bonded organic framework (HOF) enable protein integration into the HOF, regardless of the protein's physicochemical properties and residues. Furthermore, experimental validation of the self-assembly of nanoscale HOFs (nHOFs) demonstrates that the self-assembly of these nanoscale HOFs is an effective method for in situ protein encapsulation and sustained release catalyzed by enzymes in living cells. The effective encapsulation and cellular delivery of catalase using nHOFs can significantly reduce oxidative stress by degrading reactive oxygen species in neurons, thereby exerting a neuroprotective effect.

[0034] As an example, 2mM TAM and 8mM AZB can be mixed in an aqueous solution under natural environmental conditions. At this time, the hydrogen bond interaction between the amidine group of TAM and the carboxylate group of AZB causes TA-HOF to form. TA-HOF is specifically in the form of nanorods with a length of about 200nm. The size and morphology of TA-HOF are not affected by the assembly ratio between TAM and AZB. The hydrogen bond between the residues of the protein catalase and TA-HOF is further utilized to encapsulate the catalase precursor into TA-HOF. When the concentration of catalase is fixed, for example, fixed at 5mM, the particle size of Catalase@nHOFs nanoparticles is inversely proportional to the concentration of TAM and AZB, that is, the particle size can be reduced from 2 mm to 500 nm as the concentration increases.

[0035] As a possible implementation method, Catalase@nHOFs nanoparticles are adhered to the surface of the artificial cornea using tissue adhesive. Using tissue adhesive to adhere Catalase@nHOFs nanoparticles to the surface of the artificial cornea has the advantages of simple process and easy operation.

[0036] As a possible implementation, tissue adhesives include at least biomimetic recombinant type III mussel mucin, recombinant mussel mucin rMfp151, mussel proteins containing Fe3+, and mussel mucin modified with glycosylation. Catalase@nHOFs nanocoatings adhere to the artificial cornea in the same way as mussel mucin, demonstrating advantages such as high adhesion strength and excellent biocompatibility.

[0037] As a possible implementation method, the biomimetic recombinant type III mussel mucin includes a protein that constitutes the mussel byssus fiber skeleton and an Mfp-3 protein expressed in fusion with the protein. Some or all of the tyrosine residues of the Mfp-3 protein are modified into dopa groups through the action of co-expressed tyrosinase.

[0038] As a possible implementation method, the recombinant mussel mucin rMfp151 is a fusion of the Mcfp-5 mature peptide of the thick-shell mussel Mytilus coruscus and the Mefp-1 peptide of the blue mussel Mytilus edulis at both ends.

[0039] As a possible implementation method, mussel mucin is an iron-based mussel protein. Artificial cornea generally includes a PMMA material bracket and a medical metal titanium bracket. The bracket is generally sutured on the eye surface or inside the eye. The Fe in the iron-based mussel protein can be used to 3+ The metal-catechol coordination bond is constructed with Dopa to enhance the wear and corrosion resistance. It needs to be further explained that firstly, the Fe 3+ The MFP-5 solution was then dropped onto the surface of the artificial cornea using the hanging drop method to form a coating. 3 + In non-free state.

[0040] As a possible approach, glycosylated mussel mucin can be used. This modification creates more microstructures that, when combined with adherent nanoparticles, could potentially achieve long-term sustained release of the nanoparticles. Glycosylation of mussel mucin is an existing method and will not be discussed further here.

[0041] As a possible implementation, a tissue adhesive is a medical tissue adhesive based on mussel mucin and hyaluronic acid. Specifically, it is obtained by the following method: recombinant mussel mucin, specifically either fp151 or rMfp151, is obtained. The recombinant mussel mucin is then DOPA-ified using enzyme catalysis to obtain a recombinant mussel protein containing a DOPA group. Hyaluronic acid and dopamine hydrochloride are prepared, and the hyaluronic acid is oxidized to obtain aldehyde-modified hyaluronic acid. The aldehyde-modified hyaluronic acid reacts with dopamine hydrochloride via a Schiff base reaction to obtain dopamine-grafted hyaluronic acid. The recombinant mussel protein containing a DOPA group and the dopamine-grafted hyaluronic acid form a coacervate through electrostatic interaction, and multiple bioactive components, including cockroach thymosin THY3, are encapsulated in the process, forming a medical tissue adhesive with strong adhesion and bioactivity in humid or underwater environments.

[0042] As a possible implementation method, the mussel-like mucin obtained by tyrosinase oxidation of mussel-like mucin intermediates mlp353, mlp316 and mlp319 is applied to the artificial cornea provided by the embodiment of the present invention, which has higher adhesion and more uniform molecular weight distribution.

[0043] As a possible implementation, a hydroxyapatite layer is also deposited between the tissue adhesive and the artificial cornea surface. This layer of hydroxyapatite provides an antibacterial effect. Furthermore, the irregular and diverse surface structure of hydroxyapatite facilitates adhesion of the mussel adhesive, making it less susceptible to erosion or metabolism. This ensures the stability of the Catalase@nHOFs coating, allowing for the long-term release of catalase.

[0044] As a possible implementation, the lap-shear tensile bearing strength of the tissue adhesive is 0.23-0.24 MPa.

[0045] Using 2',7'-dichlorodihydrofluorescein diacetate staining and imaging as well as flow cytometry analysis, it was found that HOF-encapsulated catalase could significantly enhance the ROS level of cells, that is, it could significantly reduce cellular oxidative stress and increase cell survival rate by 60%.

[0046] Although the present invention is described herein in conjunction with various embodiments, in the process of implementing the claimed invention, those skilled in the art can understand and implement other variations of the disclosed embodiments by reviewing the disclosed content. In the specification, the word "comprising" does not exclude other components or steps, and "a" or "an" does not exclude multiple situations. A single processor or other unit can implement several functions listed in the specification. Certain measures are recorded in different embodiments, but this does not mean that these measures cannot be combined to produce good results.

[0047] Although the present invention has been described with reference to specific features and embodiments thereof, it will be apparent that various modifications and combinations thereof may be made without departing from the spirit and scope of the invention. Accordingly, this specification is merely illustrative of the present invention and is deemed to cover any and all modifications, variations, combinations or equivalents within the scope of the invention. It will be apparent that various modifications and variations of the present invention may be made by those skilled in the art without departing from the spirit and scope of the invention. Thus, the present invention is intended to include such modifications and variations as fall within the scope of the invention and its equivalents.

Claims

1. An artificial cornea with a Catalase@nHOFs nanocoating, characterized in that: The portion of the artificial cornea that contacts the eye is provided with a Catalase@nHOFs nanocoating; the Catalase@nHOFs nanocoating is prepared by encapsulating catalase in a hydrogen-bonded organic framework to form Catalase@nHOFs nanoparticles, which adhere to the surface of the artificial cornea to form the Catalase@nHOFs nanocoating; The material of artificial cornea is polymethyl methacrylate; The Catalase@nHOFs nanoparticles are adhered to the surface of the artificial cornea using tissue adhesive; The tissue adhesive at least includes biomimetic recombinant type III mussel mucin, recombinant mussel mucin rMfp151, iron-based mussel protein, mussel mucin modified by glycosylation, medical tissue adhesive based on mussel mucin and hyaluronic acid, and mussel-like mucin obtained by oxidizing mussel-like mucin intermediates mlp353, mlp316 and mlp319 with tyrosinase; The biomimetic recombinant type III mussel adhesive protein includes a protein constituting the mussel byssus fiber skeleton and an Mfp-3 protein expressed by fusion with the protein, wherein part or all of the tyrosine residues of the Mfp-3 protein are modified into dopa groups by the action of co-expressed tyrosinase; The recombinant mussel mucin rMfp151 is a mature peptide of Mcfp-5 from the thick-shelled mussel Mytilus coruscus fused with the Mefp-1 peptide from the blue mussel Mytilus edulis at both ends; A hydroxyapatite layer is also plated between the tissue adhesive and the surface of the artificial cornea.

2. The artificial cornea with Catalase@nHOFs nanocoating according to claim 1, characterized in that: The thickness of Catalase@nHOFs nanocoating is 0.5um to 50um.

3. The artificial cornea with Catalase@nHOFs nanocoating according to claim 1, characterized in that: Catalase@nHOFs nanoparticles are in the form of nanorods with a length of 200 nm.

4. The artificial cornea with Catalase@nHOFs nanocoating according to claim 1, characterized in that: The tissue adhesive has a lap-shear tensile bearing strength of 0.23 to 0.24 MPa.