Patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 and preparation method thereof

Abstract

The present invention discloses a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 and a preparation method thereof. The invention comprises the following steps: synthesizing mesoporous bioactive glass (BG) by a sol-gel method; modifying the surface of BG with amino groups using a silane coupling agent (APTES) to obtain amino-modified mesoporous bioactive glass (BG-NH2); utilizing BG-NH2 as a carrier of miR-133b, and co-incubating the two to obtain miR-133b@BG-NH2 nanoparticles; dissolving collagen by an acid dissolution method to obtain a collagen solution, adding a cross-linking agent for cross-linking; blending the collagen solution with miR-133b@BG-NH2 to obtain a mixed solution, stirring to remove bubbles, pouring the solution into a PDMS mold with a microgroove structure, and naturally air-drying the solution to form a film; immersing the film in a cross-linking agent and cross-linking it again, and air-drying the film to obtain the product. The method of the present invention is easy to implement, and the obtained artificial cornea has good biocompatibility, similar transparency to that of a natural cornea, and meets the mechanical properties and regulatable degradation properties required for corneal repair.

Description

Technical Field

[0001] The present invention relates to the technical fields of biomedical materials, tissue engineering and regenerative medicine, and in particular to a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 and a preparation method thereof. Technical Background

[0002] Blindness caused by corneal damage or disease is a common ophthalmic condition, currently treated with corneal transplants from deceased donors. However, corneal transplants are difficult to obtain, and immune rejection often leads to transplant failure. Relying solely on human corneal transplants falls far short of meeting treatment needs. To alleviate the shortage of corneal donors, there is an urgent clinical need to develop artificial corneal substitutes. Biocompatible, highly transparent, and fully biodegradable artificial corneas can protect damaged corneal tissue, guide and promote corneal regeneration, and hold the promise of alleviating the shortage of corneal donors and reducing the burden on patients.

[0003] In recent years, commercially available artificial corneas have been divided into two main categories: non-tissue-engineered artificial corneas, primarily composed of optical prisms, commonly made of materials such as poly(hydroxyethyl methacrylate), poly(methyl methacrylate), silicone gel, and glass; and peripheral stents, commonly made of ceramics, fluorocarbon polymers, hydroxyapatite, biomaterials, and polytetrafluoroethylene. Tissue-engineered artificial corneas, primarily made of biological tissues, commonly including pig and fish scales and human skin, have been found. However, these artificial corneas currently suffer from relatively poor light transmittance, biocompatibility, bioactivity, and mechanical properties, necessitating an urgent need for a qualified corneal repair material.

[0004] The existing artificial cornea made of collagen has the following disadvantages: the collagen membrane material prepared with collagen has weak mechanical strength and cannot withstand the suturing of surgical sutures and the rapid degradation of collagenase in the body. It lacks biological activity and topological structure regulation, which makes it difficult to fix the artificial cornea on the ocular surface, slows postoperative recovery, and increases surgical risks. Summary of the Invention

[0005] The present invention aims to address the shortcomings and deficiencies of existing artificial corneas by providing a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 and a method for preparing the same. The artificial cornea prepared by this method has good biocompatibility, similar transparency to a natural cornea, mechanical properties required for corneal repair, and controllable degradation properties. The patterned microgrooved structure on the surface of the artificial cornea can accelerate the healing of corneal injuries and inhibit scar formation. In addition, the miR-133b@BG-NH2 released by the artificial cornea can further inhibit scar formation, thereby better treating corneal injuries.

[0006] To achieve the above objectives, the present invention provides a technical solution: a method for preparing a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2, comprising the following steps:

[0007] 1) Surface amino modification of material A, dissolving the amino-modified material A and material B in deionized water in proportion, mixing them evenly, and incubating at room temperature to obtain nanoparticles; wherein the material A is an amino-modified mesoporous bioactive glass, referred to as BG-NH2, and the material B is miR-133b;

[0008] 2) The obtained nanoparticles and material C solution are blended in proportion, stirred to remove bubbles, poured into a patterned mold, and naturally air-dried to form a film, which is then cross-linked with a cross-linking agent and air-dried to obtain a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2; wherein, the material C is bovine Achilles tendon type I collagen.

[0009] Preferably, in step 1), the preparation of the material A comprises the following steps:

[0010] First, CTAB is dissolved in deionized water, followed by the addition of ethyl acetate and continued stirring to form uniform microemulsion droplets; then ammonia water is added dropwise; finally, TEOS, TEP, and calcium nitrate are added in sequence and continued stirring to allow the solution to react fully. The solution is then allowed to stand for aging, centrifuged, and washed to obtain a white precipitate. After vacuum drying, high-temperature calcination, grinding, and sieving, a mesoporous bioactive glass, called BG, is obtained.

[0011] Preferably, in step 1), the amino modification of material A comprises the following steps:

[0012] APTES was thoroughly mixed in n-hexane to prepare a reaction solution, and then the obtained BG was added to the reaction solution and ultrasonically dispersed to obtain a mixed solution. The mixed solution was stirred evenly and then centrifuged. The precipitate was washed with anhydrous ethanol and deionized water, and finally vacuum dried to obtain amino-modified mesoporous bioactive glass, namely BG-NH2.

[0013] Preferably, in step 1), the surface amino-modified material A and the material B are blended, incubated at room temperature, and the precipitate is collected by centrifugation to obtain miR-133b@BG-NH2 nanoparticles.

[0014] Preferably, in step 2), the preparation of the material C solution comprises the following steps:

[0015] Bovine Achilles tendon type I collagen was dissolved in a hydrochloric acid solution and stirred until completely dissolved to prepare a collagen solution with a concentration of 2.5 mg / ml to 10 mg / ml. The cross-linking agent EDC / NHS was added and stirred evenly to obtain a material C solution.

[0016] Preferably, in step 2), the concentration of miR-133b@BG-NH2 nanoparticles used is 0.3 wt% to 1 wt%.

[0017] Preferably, in step 2), the patterned mold surface has a micro-groove structure, with a groove width of 25 μm to 100 μm, a groove ridge width of 200 μm, and a groove depth of 50 μm.

[0018] Preferably, in step 2), the cross-linking agent used is EDC / NHS, and the soaking time is 24 to 48 hours.

[0019] The present invention also provides a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 prepared by the above method. The cornea has good biocompatibility and meets the transparency and water content requirements of the artificial cornea. The patterned groove structure can guide cell migration, promote corneal epithelial healing and inhibit the differentiation of corneal stromal cells into myofibroblasts; the released miR-133b can further inhibit the differentiation of corneal stromal cells into myofibroblasts and inhibit scar formation. It has the potential to accelerate corneal epithelial healing and scar-free corneal repair, thereby better treating corneal injuries.

[0020] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0021] 1. The present invention simultaneously introduces topological structure and bioactive factors to develop a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2, which can solve the problems existing in current artificial corneas to a certain extent.

[0022] 2. The artificial cornea prepared by the present invention is prepared with type I collagen as raw material. Collagen is the main component of natural cornea and has good biocompatibility. The surface has rich RGD sequences, which are conducive to the adhesion and proliferation of corneal cells.

[0023] 3. The artificial cornea prepared by the present invention is formed by self-assembly of collagen fibers layer by layer and has a transparency similar to that of a natural cornea.

[0024] 4. The artificial cornea prepared by the present invention is cross-linked by EDC / NHS, which significantly improves its mechanical properties and meets the requirements of corneal transplantation.

[0025] 5. The artificial cornea prepared by the present invention has a denser internal structure due to EDC / NHS cross-linking. Due to the influence of steric hindrance, some of the action sites of collagenase are shielded, thereby delaying the degradation of the artificial cornea under physiological conditions.

[0026] 6. The artificial cornea prepared by the present invention introduces miR-133b, which can inhibit the differentiation of corneal stromal cells into corneal fibroblasts and the expression of fibroblast-related marker genes, thereby inhibiting the formation of scar tissue during corneal repair.

[0027] 7. The present invention prepares BG-NH2 nanoparticles with positive surface charge, which bind to negatively charged miR-133b through electrostatic interaction to achieve miR-133b loading, effectively protect miR-133b, enhance its stability, and enable it to be internalized by corneal stromal cells.

[0028] 8. By incorporating miR-133b@BG-NH2 into the artificial cornea, the purpose of sustained release of miR-133b was achieved, enabling it to play a long-term role in inhibiting scar formation.

[0029] 9. The surface of the artificial cornea produced by the present invention has a micro-groove structure. The presence of the micro-groove structure can guide the migration of corneal cells and promote corneal wound healing; in addition, the micro-groove structure can also inhibit the formation of corneal scars.

[0030] 10. The artificial cornea prepared by the present invention has both a microgroove structure and miR-133b@BG-NH2. Under the combined action of the two, it accelerates the repair of corneal damage and has the potential for rapid and scar-free repair of the cornea. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Microscopic morphology of mesoporous bioactive glass before and after amino modification.

[0032] Figure 2 The comparison of the depth of the electrophoretic bands of miR-133b@BG-NH2 and miR-133b at different time points after incubation in fetal bovine serum.

[0033] Figure 3 The macromorphology and scanning electron microscopy characterization of the patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0034] Figure 4 This is a characterization diagram of the water content of the patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0035] Figure 5 The transmittance characterization diagram of the patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0036] Figure 6 Figure 2 shows the release characterization of miR-133b from patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0037] Figure 7 Figure 2 shows the cell compatibility characterization of patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0038] Figure 8 Figure 2 shows the cell migration characterization of patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0039] Figure 9 Figure 2 shows the cell differentiation characterization of patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2. DETAILED DESCRIPTION

[0040] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0041] Example 1

[0042] 1) First, 4.2g of CTAB was placed in deionized water and stirred until completely dissolved. 30mL of ethyl acetate was then added dropwise to the solution, and stirring continued for 30 minutes to form a uniform microemulsion. A certain amount of ammonia was then added dropwise to corrode the ethyl acetate, forming mesoporous channels that served as a template for subsequent reactions. Finally, 21.6mL of TEOS, 2.16mL of TEP, and 13.65g of calcium nitrate were added sequentially, with 30-minute intervals between each addition. Stirring was continued for 4 hours to allow the solution to fully react, followed by 24 hours of aging. The white precipitate was centrifuged and washed, vacuum dried, calcined in air at 650°C for 3 hours, and ground and sieved to obtain bioactive glass (BG).

[0043] 2) First, APTES was thoroughly mixed in n-hexane to prepare a reaction solution. BG was then added to the reaction solution and ultrasonically dispersed to obtain a mixed solution. The mixed solution was stirred at 60°C for 3 hours and then centrifuged. The precipitate was washed with anhydrous ethanol and deionized water, and finally vacuum-dried to obtain amino-modified bioactive glass (called BG-NH2).

[0044] 3) miR-133b was mixed with a solution of amino-modified mesoporous bioactive glass nanoparticles at a mass ratio of 32:1. After incubation at room temperature for 15 minutes, the mixture was centrifuged to obtain miR-133b@BG-NH2 nanoparticles.

[0045] 4) Type I collagen extracted from beef tendon was added to a 0.01 mol / L hydrochloric acid solution at a ratio of 2.5 mg / mL and stirred at 4°C to fully dissolve the collagen powder to form a collagen solution with a concentration of 2.5 mg / ml. Then, aqueous solutions of EDC and NHS were added (the mass ratio of EDC to NHS was 1:1, and the mass ratio of collagen to EDC was 6:1). After stirring for 4 hours, miR-133b@BG-NH2 nanoparticles were added at a concentration of 0.3 wt%. The mixture was stirred at room temperature to remove bubbles. The film was then poured onto the surface of a patterned PDMS mold (with a microgroove structure, a groove width of 100 μm, a groove ridge width of 200 μm, and a groove depth of 50 μm). After air drying, a patterned collagen film was obtained. The collagen membrane was then immersed in a 0.1 M EDC / NHS solution (i.e., cross-linking agent) for 48 h and air-dried to obtain a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2.

[0046] The SEM and TEM images of the mesoporous bioactive glass (called BG) prepared by the sol-gel method and the bioactive glass modified with amino groups on the surface of the silane coupling agent APTES (called BG-NH2) are shown in Figure 2. Figure 1 As shown in the figure, the average particle size of both is around 150nm, and the surface morphology does not change significantly before and after amino modification. The positive charge on the BG-NH2 surface and the negatively charged miR-133b achieve miR-133b loading through electrostatic interaction. Figure 2 The following is a comparison of the depth of the electrophoretic bands of miR-133b@BG-NH2 and miR-133b incubated in fetal bovine serum at different time points. It can be seen that naked miRNA (free miRNA without a carrier) is easily degraded by RNase enzymes in physiological environments, thereby losing its biological activity. The use of BG-NH2 as a nucleic acid carrier improves the stability of miR-133b, enabling it to exert its effect long-term at the site of injury. Because collagen molecules can be evenly dispersed in hydrochloric acid solution, as the solution continues to evaporate, the self-assembly between molecules makes the internal structure of the collagen-based corneal regeneration and repair material layered and orderly. Figure 3 The following are macroscopic and scanning electron microscopic images of the collagen-based artificial cornea prepared in this example. The collagen-based corneal repair material exhibits good transparency and a distinct surface groove structure. During surgical suturing, insufficient suture tensile strength often leads to surgical failure. Because pure Col membranes cannot meet clinical suturing requirements, EDC / NHS chemical crosslinking is used to enhance their mechanical properties and address the problem of suture tearing during surgery. Figure 4This is a characterization diagram of the water content of the collagen-based artificial cornea prepared in this example. It can be seen from the figure that the effects of the microgroove structure and the introduction of miR-133b@BG-NH2 on the water content are negligible. Figure 5 This is a characterization of the transmittance of the collagen-based artificial cornea prepared in this embodiment in the visible light region. As shown in the figure, the introduction of the microgroove structure causes a slight decrease in its transmittance in the visible light region, but it still meets the requirements of corneal transplantation. This may be due to the light refraction caused by the surface groove structure. Figure 6 This is the release curve of miR-133b from the collagen-based artificial cornea prepared in this example. The results show that because miR-133b is protected by BG-NH2 and doped into the artificial cornea, it can be released in a burst to reach an effective concentration within a short period of time, followed by a long-term sustained release, which may give the collagen-based artificial cornea a longer-lasting ability to inhibit corneal scarring. Figure 7 This is a characterization diagram of the cell compatibility of the artificial cornea prepared in this example. It can be seen from the figure that the introduction of the groove structure and miR-133b@BG-NH2 has no significant effect on the cell proliferation on the artificial cornea. As time goes by, human corneal stromal cells can obviously adhere to and proliferate on the collagen-based corneal regeneration and repair material. Figure 8 This is a characterization diagram of the cell compatibility of the artificial cornea prepared in this example. It can be seen from the cell migration results that 24 hours after the start of the scratch experiment, the scratches on the patterned collagen film with a groove structure almost completely disappeared, while obvious scratches still existed in the flat collagen group, indicating that the microgroove structure can significantly promote cell migration, which is beneficial to corneal wound healing. Figure 9 This is a cell differentiation characterization diagram of the artificial cornea prepared in this example. From the PCR results in the figure, it can be seen that the groove structure can inhibit the expression of corneal fibroblast marker genes, and the addition of miR-133b further inhibits the differentiation of corneal stromal cells into myofibroblasts, indicating that the artificial cornea prepared in this example has the function of inhibiting scar formation and has the potential to achieve scarless corneal repair.

[0047] Example 2

[0048] The difference from Example 1 is that the concentration of the collagen solution in step 4) is 10 mg / ml.

[0049] Example 3

[0050] The difference from Example 1 is that in step 4), the concentration of miR-133b@BG-NH2 nanoparticles used is 1 wt%.

[0051] Example 4

[0052] The difference from Example 1 is that the micro-groove structure on the surface of the PDMS mold used in step 4) has a groove width of 25 μm, a groove ridge width of 200 μm, and a groove depth of 50 μm.

[0053] Example 5

[0054] The difference from Example 1 is that in step 4), a 0.1 M EDC / NHS solution was used to soak the collagen membrane for 24 h.

[0055] Comparative Example 1

[0056] The difference from Example 1 is that the PDMS mold used in step 4) is a smooth mold without a microgroove structure on the surface, no miR-133b@BG-NH2 nanoparticles are added to the collagen solution, and the film is not cross-linked with EDC / NHS after formation.

[0057] Comparative Example 2

[0058] The difference from Example 1 is that in step 4), miR-133b@BG-NH2 nanoparticles were not added to the collagen solution, and the film was not cross-linked with EDC / NHS after formation.

[0059] Table 1

[0060]

[0061] As can be seen from Table 1 above, Example 2 shows that the collagen concentration in the precursor solution has a significant effect on the mechanical strength of the artificial cornea. Increasing the collagen concentration in the precursor solution improves its mechanical strength due to the increased cross-linking density. Example 3 shows that within a certain concentration range, the effect of the artificial cornea in inhibiting the expression of myofibroblast-related marker genes is enhanced due to the increase in the miR-133b@BG-NH2 content. As can be seen from Example 4, the microgroove structure has a significant effect on the regulation of corneal cell behavior. Corneal stromal cells respond differently to microgroove structures of different sizes. Compared with microgrooves with a width of 25 μm, microgrooves with a width of 100 μm have a stronger inhibitory effect on the transformation of corneal stromal cells into myofibroblasts. Example 5 shows that the cross-linking time of the artificial cornea in EDC / NHS will significantly affect its mechanical properties and transparency. The longer the cross-linking time, the denser the cross-linking between the collagen fibers, which leads to improved mechanical properties. Comparative Example 1 shows that EDC / NHS cross-linking significantly improves the mechanical properties of the artificial cornea. Furthermore, the groove structure significantly influences cell behavior, promoting corneal stromal cell migration and inhibiting the transformation of corneal stromal cells into myofibroblasts. Comparative Example 2 and Example 1 demonstrate that the microgroove structure and the presence of miR-133b@BG-NH2 synergistically downregulate the expression of myofibroblast marker genes, inhibiting the differentiation of corneal stromal cells into myofibroblasts, reducing scar formation during corneal repair and demonstrating the potential for scarless corneal repair.

[0062] The embodiments of the present invention are merely examples for the purpose of illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above embodiments, and it is not necessary or possible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims.

Claims

1. A method for preparing a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2, characterized in that: The following steps are involved: 1) Surface amino modification of material A, dissolving the amino-modified material A and material B in deionized water in proportion, mixing them evenly, and incubating at room temperature to obtain nanoparticles; wherein material A is amino-modified mesoporous bioactive glass, referred to as BG-NH2, and material B is miR-133b; 2) The obtained nanoparticles and material C solution were blended in proportion, stirred to remove bubbles, poured into a patterned mold, and naturally air-dried to form a film. The film was then cross-linked with a cross-linker and air-dried to obtain a patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2; wherein, the material C was bovine Achilles tendon type I collagen; the miR-133b@BG-NH2 nanoparticles were used at a concentration of 0.3wt% to 1wt%; and the surface of the patterned mold had a microgroove structure with a groove width of 25µm to 100µm, a groove ridge width of 200µm, and a groove depth of 50µm.

2. The preparation method according to claim 1, wherein: In step 1), the preparation of the material A comprises the following steps: First, CTAB is dissolved in deionized water, followed by the addition of ethyl acetate and continued stirring to form uniform microemulsion droplets; then ammonia water is added dropwise; finally, TEOS, TEP, and calcium nitrate are added in sequence and continued stirring to allow the solution to react fully. The solution is then allowed to stand for aging, centrifuged, and washed to obtain a white precipitate. After vacuum drying, high-temperature calcination, grinding, and sieving, a mesoporous bioactive glass, called BG, is obtained.

3. The preparation method according to claim 2, wherein: In step 1), the amino modification of material A comprises the following steps: APTES was thoroughly mixed in n-hexane to prepare a reaction solution, and then the obtained BG was added to the reaction solution and ultrasonically dispersed to obtain a mixed solution. The mixed solution was stirred evenly and then centrifuged. The precipitate was washed with anhydrous ethanol and deionized water, and finally vacuum dried to obtain amino-modified mesoporous bioactive glass, namely BG-NH2.

4. The preparation method according to claim 3, wherein: In step 1), the surface amino-modified material A and material B are blended, incubated at room temperature, and the precipitate is collected by centrifugation to obtain miR-133b@BG-NH2 nanoparticles.

5. The preparation method according to claim 4, characterized in that: In step 2), the preparation of the material C solution includes the following steps: Dissolve bovine Achilles tendon type I collagen in hydrochloric acid solution and stir until completely dissolved to prepare a collagen solution with a concentration of 2.5 mg / ml to 10 mg / ml. Add the cross-linking agent EDC / NHS and stir evenly to obtain Material C solution.

6. The preparation method according to claim 5, characterized in that: In step 2), the cross-linking agent used is EDC / NHS, and the immersion time is 24h~48h.

7. A patterned collagen-based artificial cornea loaded with miR-133b@BG-NH2 prepared by the preparation method according to any one of claims 1 to 6, wherein the cornea has good biocompatibility and meets the transparency and water content requirements of the artificial cornea. The patterned groove structure can guide cell migration, promote corneal epithelial healing and inhibit the differentiation of corneal stromal cells into myofibroblasts; the released miR-133b can further inhibit the differentiation of corneal stromal cells into myofibroblasts and inhibit scar formation, and has the potential to accelerate corneal epithelial healing and scar-free corneal repair, thereby better treating corneal damage.

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