Artificial cornea applying Catalase (at) nHOFs nanoparticles

Abstract

The invention discloses an artificial cornea applying Catalase (at) nHOFs nanoparticles, and belongs to the technical field of in-vivo prosthesis implantation. Frequent administration is not needed, long-acting ROS resistance can be achieved, and in addition, an existing artificial cornea structure does not need to be adjusted or changed. The part in the artificial cornea contact eye is provided with a Catalase (at) nHOFs nano coating; the Catalase (at) nHOFs nano coating is formed by encapsulating catalase by a hydrogen bond organic framework to prepare Catalase (at) nHOFs nano particles, and then the Catalase (at) nHOFs nano particles are adhered to the surface of the artificial cornea to form the Catalase (at) nHOFs nano coating.

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] Artificial cornea is the last and only hope for patients with end-stage corneal blindness to regain their sight. However, artificial cornea implantation often induces a persistent foreign body reaction, with abnormal increase in reactive oxygen species (ROS) as the main pathological manifestation, leading to complications such as sterile endophthalmitis and optic nerve damage after surgery, reducing long-term efficacy. Therefore, how to inhibit the pathological increase in ROS after artificial cornea surgery is the key to maintaining long-term efficacy.

[0003] The development of anti-ROS drugs has always been an international hot spot and difficulty. Currently, there are no anti-ROS drugs in ophthalmology. The drugs under development (such as hydrogen water) have poor stability, limited action time, and need 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 vitreous cavity injection. 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 fight ROS for a long time 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 can catalyze the decomposition of hydrogen peroxide into oxygen and water, and is the most powerful tool for organisms to resist ROS. However, catalase is easily denatured and its activity is reduced in pathological environments.

[0006] A long-acting anti-ROS drug based on HOF was prepared and coated on an artificial cornea (PMMA material), and an artificial cornea with long-term anti-ROS effect was developed. 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 resistance to 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 nano-coating, wherein the portion of the artificial cornea contacting the inside of the eye has the Catalase@nHOFs nano-coating; the Catalase@nHOFs nano-coating 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 nano-coating.

[0010] As a possible implementation method, the thickness of the Catalase@nHOFs nano-coating 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 the hydrogen bonding interaction between them is used to achieve self-assembly;

[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 at least includes bionic 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 bionic recombinant type III mussel mucin includes a protein that constitutes the mussel byssus fiber skeleton and an Mfp-3 protein expressed by fusion with the protein, and 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 Mefp-1 peptide of the blue mussel Mytilus edulis with the Mcfp-5 mature peptide of the thick-shelled mussel Mytilus coruscus 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 lap-shear tensile bearing strength of the tissue adhesive is 0.23-0.24 MPa.

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

[0022] 1. The Catalase@nHOFs nanocoating is adhered to the part of the artificial cornea that contacts the eye. The raw material of the Catalase@nHOFs nanocoating is prepared by encapsulating catalase in a hydrogen-bonded organic framework to form Catalase@nHOFs nanoparticles. That is, catalase is encapsulated in a hydrogen-bonded organic framework (HOF). The protective environment provided by HOF can effectively maintain the activity of catalase, and the Catalase@nHOFs nanoparticles can be efficiently taken up by cells, and the enzyme catalysis reaction can be carried out stably in the cell for a long time, thereby reducing ROS in a long-term manner. In addition, Catalase@nHOFs nanoparticles can form hydrogen bonds with solid phase materials such as polymethyl methacrylate (PMMA), and stably combine to form an implantable enzyme-catalyzed anti-ROS drug. Polymethyl methacrylate (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 frequent drug administration and low drug delivery efficiency in existing artificial corneas.

[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 resistance and corrosion resistance of artificial corneas. For example, mussel mucins can be glycosylated to obtain more microstructures, and then Catalase@nHOFs nanoparticles can be adhered to the artificial cornea to obtain long-term sustained release of Catalase@nHOFs nanoparticles, and the adhesion strength is higher.

[0024] 3. The present invention also plates hydroxyapatite between the mussel mucin and the surface of the artificial cornea (hydroxyapatite is plated on the side of the lens column and the bracket). The irregular and diverse surface structure of hydroxyapatite makes the mussel mucin easier to adhere, that is, it is not easily washed away or metabolized, ensuring the stability of the Catalase@nHOFs coating, thereby allowing 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 can significantly reduce the ROS level of cells, that is, it can significantly reduce cell oxidative stress, and the cell survival rate can be increased by 60%. Cell experiments show that catalase encapsulated by HOF has no degradation trend, and it is speculated to be effective for a long time. DETAILED DESCRIPTION

[0026] In order to clearly describe the technical solutions of the embodiments of the present invention, in the embodiments of the present invention, words such as "first" and "second" are used to distinguish the same items or similar items with basically the same functions and effects. For example, the first threshold and the second threshold are only used to distinguish different thresholds, and their order is not limited. Those skilled in the art can understand that words such as "first" and "second" do not limit the quantity and execution order, and words such as "first" and "second" do not necessarily limit them to be 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 interpreted as being more preferred or more advantageous than other embodiments or designs. Specifically, the use of words such as "exemplary" or "for example" is intended to present related concepts in a specific way.

[0028] In the present invention, "at least one" means one or more, and "plurality" means 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 mean: A exists alone, A and B exist at the same time, and B exists alone, where A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items. For example, at least one of a, b or c can mean: 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 nano-coating, wherein the portion of the artificial cornea contacting the inside of the eye has the Catalase@nHOFs nano-coating; the Catalase@nHOFs nano-coating 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 nano-coating.

[0031] Catalase@nHOFs nanocoating is adhered to the part of the artificial cornea that contacts the eye. The raw material of the Catalase@nHOFs nanocoating is Catalase@nHOFs nanoparticles prepared by encapsulating catalase in a hydrogen-bonded organic framework. That is, catalase is encapsulated in a hydrogen-bonded organic framework (HOF). The protective environment provided by HOF can effectively maintain the activity of catalase, and Catalase@nHOFs nanoparticles can be efficiently taken up by cells, and the enzyme catalysis reaction can be carried out stably in the cell for a long time, thereby reducing ROS in a long-term manner. In addition, Catalase@nHOFs nanoparticles can form hydrogen bonds with solid phase materials such as polymethyl methacrylate (PMMA), and stably combine to form implantable enzyme-catalyzed anti-ROS drugs. Polymethyl methacrylate (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 frequent drug administration and low drug delivery efficiency in existing artificial corneas, and can achieve long-term anti-ROS.

[0032] As a possible implementation, the thickness of the Catalase@nHOFs nanocoating is 0.5um to 50um, specifically 0.5um, 1.0um, 5um, 10um, 15um, 20um, 25um, 30um, 35um, 40um, 45um or 50um. That is, the thickness of the nanocoating can be specifically determined according to the patient's eye conditions. For example, patients who need to take the drug for a long time can choose a thicker Catalase@nHOFs nanocoating, and vice versa, a thinner Catalase@nHOFs nanocoating can be chosen.

[0033] As a possible implementation method, hydrogen-bonded organic frameworks are used to encapsulate catalase to prepare Catalase@nHOFs nanoparticles, which specifically include the following steps: tetramethane (TAM) and azophthalate (AZB) are mixed in an aqueous solution, and the hydrogen bonding interaction between the two is used to achieve self-assembly; catalase is configured 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. Studies have shown that regardless of the physicochemical properties and residues of the protein, the hydrogen bonding interaction between the protein residues and the building blocks of the hydrogen-bonded organic framework (HOF) enables the protein to be integrated into the HOF. The self-assembly of nanoscale HOFs (nHOFs) was further verified experimentally as an effective method for in situ protein encapsulation and sustained release of enzyme-catalyzed proteins in living cells. The effective encapsulation and cellular delivery of catalase using nHOFs can significantly reduce oxidative stress by degrading the reactive oxygen species of nerve cells, thereby playing a neuroprotective role.

[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 forms TA-HOF. 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 bonds between the residues of the protein catalase and TA-HOF are 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 nanometers as the concentration increases.

[0035] As a possible implementation method, Catalase@nHOFs nanoparticles are adhered to the surface of the artificial cornea using tissue adhesives. Using tissue adhesives 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 method, tissue adhesives at least include biomimetic recombinant type III mussel mucin, recombinant mussel mucin rMfp151, mussel protein containing Fe3+, mussel mucin modified by glycosylation, etc. Catalase@nHOFs nanocoating adheres to the artificial cornea in the manner of mussel mucin, and has the advantages of high adhesion strength and good biocompatibility.

[0037] As a possible implementation method, the bionic recombinant type III mussel mucin includes a protein that constitutes the mussel byssus fiber skeleton and an Mfp-3 protein expressed by 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 the co-expressed tyrosinase.

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

[0039] As a possible implementation method, mussel mucin is an iron-based mussel protein. Artificial corneas generally include a PMMA stent and a medical titanium stent. The stent 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 with Dopa is used to enhance the wear and corrosion resistance. 3+ The MFP-5 solution is 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 implementation method, mussel mucin modified by glycosylation is used. After glycosylation modification, more microstructures are formed, and with the attached nanoparticles, long-term sustained release of nanoparticles may be obtained. Glycosylation modification of mussel mucin is an existing method and will not be described in detail here.

[0041] As a possible implementation method, the tissue adhesive is a medical tissue adhesive based on mussel mucin and hyaluronic acid. It is obtained specifically by the following method: recombinant mussel mucin is obtained, which can be any one of fp151 or rMfp151. Then, the DOPA of the recombinant mussel mucin is realized by enzyme catalysis to obtain a recombinant mussel protein containing a DOPA group. Hyaluronic acid and dopamine hydrochloride are configured, and the hyaluronic acid is oxidized to obtain aldehyde-modified hyaluronic acid. The aldehyde-modified hyaluronic acid and dopamine hydrochloride are reacted by Schiff base to obtain dopamine-grafted hyaluronic acid. The recombinant mussel protein containing the DOPA group and the dopamine-grafted hyaluronic acid are formed into a coacervate through electrostatic interaction, and a variety of bioactive ingredients including cockroach thymosin THY3 are encapsulated in the process to form a medical tissue adhesive with strong adhesion and bioactivity in a humid or underwater environment.

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

[0043] As a possible implementation method, a hydroxyapatite layer is also plated between the tissue adhesive and the surface of the artificial cornea. In the present invention, hydroxyapatite is also plated between the mussel mucin and the surface of the artificial cornea, which can play an antibacterial role on the one hand, and on the other hand, the irregular and diverse surface structure of hydroxyapatite makes the mussel mucin easier to adhere, that is, it is not easy to be washed away or metabolized, ensuring the stability of the Catalase@nHOFs coating, thereby allowing 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 may understand and implement other variations of the disclosed embodiments by viewing the disclosed content. In the specification, the word "comprising" does not exclude other components or steps, and "one" 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 in conjunction with specific features and embodiments thereof, it is apparent that various modifications and combinations may be made thereto without departing from the spirit and scope of the present invention. Accordingly, this specification is merely an exemplary illustration of the present invention and is deemed to have covered any and all modifications, variations, combinations or equivalents within the scope of the present invention. Obviously, those skilled in the art may make various modifications and variations to the present invention without departing from the spirit and scope of the present invention. Thus, the present invention is intended to include such modifications and variations if they fall within the scope of the present invention and its equivalents.

Claims

1. An artificial cornea with a Catalase@nHOFs nanocoating, characterized in that: The part of the artificial cornea that contacts the inside of the eye has 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.

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

3. The artificial cornea with Catalase@nHOFs nano-coating according to claim 1, characterized in that: The hydrogen-bonded organic framework encapsulates catalase to prepare Catalase@nHOFs nanoparticles, which specifically includes the following steps: Tetramethyleneimine and azophthalate are mixed in aqueous solution, and the hydrogen bonding interaction between them is used to achieve self-assembly; 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.

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

5. The artificial cornea with Catalase@nHOFs nano-coating according to claim 1, characterized in that: The Catalase@nHOFs nanoparticles are adhered to the surface of the artificial cornea using tissue adhesive.

6. The artificial cornea with Catalase@nHOFs nano-coating according to claim 5, characterized in that: The tissue adhesive at least includes bionic 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 oxidizing mussel mucin intermediates mlp353, mlp316 and mlp319 with tyrosinase.

7. The artificial cornea with Catalase@nHOFs nano-coating according to claim 6, characterized in that: The biomimetic recombinant type III mussel adhesive protein comprises a protein constituting the skeleton of mussel byssus fiber and an Mfp-3 protein fused 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 the co-expressed tyrosinase; The recombinant mussel mucin rMfp151 is a peptide fragment of Mefp-1 from blue mussel Mytilus edulis fused to the Mcfp-5 mature peptide from thick-shell mussel Mytilus coruscus at both ends.

8. The artificial cornea with Catalase@nHOFs nano-coating according to claim 5, characterized in that: A hydroxyapatite layer is also plated between the tissue adhesive and the surface of the artificial cornea.

9. The artificial cornea with Catalase@nHOFs nano-coating according to claim 5, characterized in that: The lap-shear tensile bearing strength of the tissue adhesive is 0.23-0.24 MPa.