Antifungal corneal stroma lens and preparation method thereof

Abstract

The invention discloses an antifungal corneal stroma lens and a preparation method thereof, and relates to the technical field of tissue engineering, and the preparation method of the antifungal corneal stroma lens comprises the steps that corneal tissue taken out through femtosecond laser small incision corneal stroma lens extraction operation is obtained, and the central thickness of the corneal tissue is 80-150 microns; cleaning and disinfecting the cornea tissue, and performing decellularization treatment through a decellularization agent to obtain a decellularized cornea tissue; and placing the acellular cornea tissue in a voriconazole solution so that voriconazole is loaded on the acellular cornea tissue to obtain the antifungal cornea stroma lens. The invention provides the antifungal corneal stroma lens, and the bioavailability of the fungal keratitis eye medicine is improved.

Description

Technical Field

[0001] The present application relates to the field of tissue engineering technology, and in particular to an antifungal corneal stromal lens and a preparation method thereof. Background Art

[0002] Fungal keratitis is a serious corneal disease caused by a fungal infection that can lead to vision loss or even blindness. This infection is more common worldwide, particularly in tropical and subtropical regions due to hot and humid environmental conditions.

[0003] Conventional treatment for fungal keratitis primarily relies on topical eye drops, such as those containing antifungal drugs, applied directly to the infected area. However, due to the unique structure of the cornea and the eye's protective mechanisms, drug retention on the surface of the eye and in the conjunctiva is extremely limited, resulting in low drug bioavailability and limiting its effectiveness in treating deep corneal infections.

[0004] The above content is only used to assist in understanding the technical solution of this application and does not constitute an admission that the above content is prior art. Summary of the Invention

[0005] The main purpose of this application is to provide an antifungal corneal stromal lens and a preparation method thereof, aiming to provide an antifungal corneal stromal lens and to improve the bioavailability of ophthalmic drugs for fungal keratitis.

[0006] To achieve the above objectives, the present invention provides a method for preparing an antifungal corneal stromal lens, the method comprising:

[0007] Obtaining corneal tissue removed by femtosecond laser small incision corneal lenticule extraction, wherein the central thickness of the corneal tissue is 80 to 150 μm;

[0008] Cleaning and disinfecting the corneal tissue, and performing decellularization treatment with a decellularizing agent to obtain decellularized corneal tissue;

[0009] The decellularized corneal tissue is placed in a voriconazole solution so that the voriconazole is loaded on the decellularized corneal tissue, thereby obtaining an antifungal corneal stroma lens.

[0010] In one embodiment, the concentration of voriconazole in the voriconazole solution is 0.5-2 mg / mL.

[0011] In one embodiment, the soaking time in the voriconazole solution is 4 to 8 hours.

[0012] In one embodiment, before the step of decellularizing the corneal tissue using a decellularizing agent, the method further comprises:

[0013] The corneal tissue is immersed in a 1.5-2M sodium chloride solution for 24-48 hours.

[0014] In one embodiment, the decellularization agent is selected from a mixture of 5-10 U / mL deoxyribonuclease and 50-100 ug / mL ribonuclease.

[0015] In one embodiment, the decellularization treatment lasts for 24 to 72 hours.

[0016] In one embodiment, the step of cleaning and disinfecting the corneal tissue comprises:

[0017] Washing the corneal tissue with a buffer solution;

[0018] Sterilize by soaking in a mixed solution of penicillin and streptomycin.

[0019] In one embodiment, the buffer solution is washed 3 to 4 times, and each washing time is 15 to 20 minutes.

[0020] In one embodiment, the duration of the immersion disinfection is 1 to 5 hours.

[0021] In addition, to achieve the above-mentioned purpose, the embodiments of the present application also provide an antifungal corneal stromal lens, which is prepared by the method described above.

[0022] One or more technical solutions proposed in this application have at least the following technical effects: by using the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens removal (SMILE procedure) as tissue material, it retains the normal tissue structure of the cornea. The obtained corneal tissue is cleaned and disinfected, and then decellularized by a decellularizing agent to remove the cellular components of the corneal tissue and reduce immunogenicity; the decellularized corneal tissue does not produce significant changes in biochemical properties such as tissue expansion rate, in vitro degradation rate, biomechanics, and transmittance, and the tissue structure of the corneal stromal lens is retained to the maximum extent. The cell voids generated after decellularization can load more drugs to achieve drug loading. At the same time, the decellularized corneal tissue can maintain good stability and functionality as a corneal material after implantation into the eye, so it can be used as an ideal drug sustained-release carrier to improve the bioavailability of the drug. Further, the decellularized corneal tissue is placed in a voriconazole solution to achieve the loading of voriconazole in the corneal tissue to produce an antifungal corneal stromal lens. Because corneal tissue itself has good light transmittance, the resulting antifungal corneal stromal lens does not significantly affect the patient's vision after implantation, ensuring the patient's visual quality and quality of life. Voriconazole can inhibit the activity of key enzymes in fungal cell membranes, which play a key role in the biosynthesis of ergosterol in fungal cell membranes. Therefore, by inhibiting the activity of this enzyme, voriconazole can block the synthesis pathway of ergosterol, an important component of fungal cell membranes. Blocked ergosterol synthesis leads to loss of cell membrane structure and function, further damaging the integrity of fungal cells and ultimately causing fungal death, achieving an antifungal effect. The corneal stromal lens loaded with voriconazole can be used as a drug sustained-release system to improve the bioavailability of ocular drugs for fungal keratitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a flow chart of the method for preparing the antifungal corneal stromal lens according to the embodiment of the present application;

[0024] Figure 2 Schematic diagram of the results of HE staining and DAPI staining of Example 1 and Comparative Example 1 of the present application;

[0025] Figure 3 Schematic diagram of light transmittance test results of Example 1 and Comparative Example 1 of the present application;

[0026] Figure 4 Schematic diagram of the in vitro degradation test results of Example 1 and Comparative Example 1 of the present application;

[0027] Figure 5 Schematic diagram of the transmission electron microscope test results of Example 1 and Comparative Example 1 of the present application.

[0028] The realization of the objectives, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0029] To make the purpose, technical solutions and advantages of the examples of the present application clearer, the technical solutions in the examples of the present application will be described clearly and completely below. Where specific conditions are not specified in the examples, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] Below, embodiments of the method for recovering lithium from used lithium iron phosphate batteries of the present application are described in detail, with appropriate reference to the accompanying drawings. However, unnecessary detailed descriptions may be omitted. For example, detailed descriptions of well-known matters and repeated descriptions of substantially identical structures may be omitted. This is to avoid unnecessary length in the following description and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided to enable those skilled in the art to fully understand the present application and are not intended to limit the subject matter recited in the claims.

[0031] The "ranges" disclosed herein are defined in terms of lower and upper limits, where a given range is defined by selecting a lower limit and an upper limit, and the selected lower and upper limits define the boundaries of the particular range. Ranges defined in this manner can be inclusive or exclusive of the end values ​​and can be combined arbitrarily, i.e., any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60 to 120 and 80 to 110 are listed for a particular parameter, it is understood that ranges of 60 to 110 and 80 to 120 are also contemplated. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, the following ranges are all contemplated: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4, and 2 to 5. In this application, unless otherwise indicated, the numerical range "a to b" is a shorthand representation of any combination of real numbers between a and b, where a and b are both real numbers. For example, a numerical range of "0-5" indicates that all real numbers between "0-5" are listed herein, and "0-5" is simply an abbreviation for these numerical combinations. Furthermore, when a parameter is expressed as an integer ≥ 2, this is equivalent to disclosing that the parameter is, for example, an integer of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0032] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0033] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0034] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may further include step (c), indicating that step (c) may be added to the method in any order, for example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0035] Unless otherwise specified, the terms "include" and "comprising" used in this application may be open-ended or closed-ended. For example, "include" and "comprising" may mean that other components not listed may also be included or that only the listed components are included.

[0036] Unless otherwise specified, the term "or" is used in this application to be inclusive. For example, the phrase "A or B" means "A, B, or both A and B." More specifically, the condition "A or B" is satisfied if any of the following conditions are met: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0037] To make the above-mentioned objects, features and advantages of the present application more clearly understood, the technical solutions of the present application are further described below with reference to the accompanying drawings and embodiments. However, the present application is not limited to the embodiments listed, but also includes any other known modifications within the scope of the rights claimed in the present application.

[0038] The term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present application. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0039] In order to better understand the technical solution of the present application, a detailed description will be given below in conjunction with the accompanying drawings and specific implementation methods.

[0040] Conventional treatment for fungal keratitis primarily involves topical eye drops, for example, antifungal eye drops applied directly to the site of infection. However, due to the unique structure of the cornea and the eye's protective mechanisms, the amount of drug retained on the surface of the eyeball and conjunctiva is extremely limited, resulting in low drug bioavailability and limiting the therapeutic effect on deep corneal infections. Ocular surface drug delivery systems can effectively improve drug bioavailability. Conventional ocular surface drug delivery systems primarily include drug-eluting amniotic membrane and drug-eluting contact lenses. Drug-eluting amniotic membranes are used to treat ocular surface diseases through amniotic membrane transplantation or amniotic membrane coverage, but the poor transparency of the amniotic membrane can lead to problems such as decreased vision. Drug-eluting contact lenses, on the other hand, become hydrated after drug loading, leading to decreased light transmittance, oxygen permeability, and biocompatibility, which can in turn cause other eye problems.

[0041] The present application provides a solution, by using the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens removal (SMILE procedure) as tissue material, which retains the normal tissue structure of the cornea. The obtained corneal tissue is cleaned and disinfected, and then decellularized by a decellularizing agent to remove the cellular components of the corneal tissue and reduce immunogenicity; the corneal tissue after decellularization does not produce significant changes in biochemical properties such as tissue expansion rate, in vitro degradation rate, biomechanics, and light transmittance, and the tissue structure of the corneal stromal lens is retained to the greatest extent. The cell voids generated after decellularization can load more drugs to achieve drug loading, and the decellularized corneal tissue can maintain good stability and functionality after being implanted into the eye as a corneal material, so it can be used as an ideal drug sustained-release carrier to improve the bioavailability of the drug. Further, the decellularized corneal tissue is placed in a voriconazole solution to achieve the loading of voriconazole in the corneal tissue to prepare an antifungal corneal stromal lens. Because corneal tissue itself has good light transmittance, the resulting antifungal corneal stromal lens does not significantly affect the patient's vision after implantation, ensuring the patient's visual quality and quality of life. Voriconazole can inhibit the activity of key enzymes in fungal cell membranes, which play a key role in the biosynthesis of ergosterol in fungal cell membranes. Therefore, by inhibiting the activity of this enzyme, voriconazole can block the synthesis pathway of ergosterol, an important component of fungal cell membranes. Blocked ergosterol synthesis leads to loss of cell membrane structure and function, further damaging the integrity of fungal cells and ultimately causing fungal death, achieving an antifungal effect. The corneal stromal lens loaded with voriconazole can be used as a drug sustained-release system to improve the bioavailability of ocular drugs for fungal keratitis.

[0042] The first aspect of the present invention provides a method for preparing an antifungal corneal stromal lens, referring to Figure 1 , including the following steps:

[0043] Step S10, obtaining corneal tissue removed by femtosecond laser small incision corneal lenticule extraction, wherein the central thickness of the corneal tissue is 80 to 150 μm;

[0044] In one feasible embodiment, corneal tissue removed via femtosecond laser small-incision lenticule extraction (SMILE) is used as the corneal tissue. Because SMILE minimizes the severing of collagen fibers within the corneal stroma, it preserves more of the normal corneal structure and morphology, providing more intact and biomechanically stable corneal tissue. The corneal tissue can be stored in anhydrous glycerol for subsequent use.

[0045] In one feasible embodiment, the central thickness of the corneal tissue is 80 to 150 μm; for example, 80 μm, 85 μm, 90 μm, 95 μm, 100 μm, 105 μm, 110 μm, 115 μm, 120 μm, 125 μm, 130 μm, 135 μm, 140 μm, 145 μm, 150 μm, etc. If the central thickness of the corneal tissue is too small, the amount of drug that can be loaded will be limited, which may result in insufficient drug release and fail to meet treatment needs. Therefore, in the embodiment of the present application, the central thickness of the corneal tissue is determined to be 80 to 150 μm.

[0046] Step S20, cleaning and disinfecting the corneal tissue, and performing decellularization treatment with a decellularizing agent to obtain decellularized corneal tissue;

[0047] In one feasible embodiment, corneal tissue is cleaned and disinfected, then decellularized using a decellularizing agent to produce decellularized corneal tissue. Decellularization effectively removes cellular components from the corneal tissue, reducing immunogenicity and lowering the rejection rate of corneal stromal lenses. Furthermore, the resulting cellular voids can accommodate more drugs, achieving drug loading. Furthermore, decellularized corneal tissue maintains good stability and functionality after intraocular implantation, making it an ideal sustained-release drug carrier to enhance drug bioavailability.

[0048] In one feasible embodiment, the apparatus is washed with a buffer solution 3 to 4 times, each time for 15 to 20 minutes; disinfected by soaking in a mixed solution containing 0.01 to 0.07 mg / mL penicillin and 0.05 to 0.15 mg / mL streptomycin for 1 to 5 hours, and then rinsed with a buffer solution, wherein the buffer solution includes PBS and / or BBS.

[0049] In one feasible embodiment, before step S20, the step of performing decellularization with a decellularizing agent, the method further includes:

[0050] Step S21: soak the corneal tissue in a 1.5-2M sodium chloride solution for 24-48 hours.

[0051] In one feasible embodiment, the corneal tissue is immersed in a sodium chloride solution; the high osmotic pressure of the sodium chloride solution is used to allow the water in the cells to penetrate outside the cells, causing the cells to dehydrate and rupture, which is beneficial for removing the cellular components in the tissue. In addition to rupturing the cells, the sodium chloride solution can also dissolve non-collagenous proteins, thereby further purifying the collagen content in the scaffold material. The molar concentration of the sodium chloride solution is 1.5 to 2M; for example, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M, 2M, etc. When the concentration of the sodium chloride solution is too high, it may cause changes in tissue structure and loss of function; and a sodium chloride solution with too low a concentration may also result in poor decellularization treatment effect. Therefore, the embodiment of the present application determines that the molar concentration of the sodium chloride solution is 1.5 to 2M.

[0052] In one feasible embodiment, the soaking time in the sodium chloride solution is 24 to 48 hours; for example, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 ​​hours, etc.

[0053] In one feasible embodiment, after the step of soaking the corneal tissue in a 1.5-2M sodium chloride solution for 24-48 hours in step S21, the method further includes: rinsing with a buffer solution for 48-96 hours, wherein the buffer solution is uniformly shaken on a shaker at 100 r / h and the buffer solution is replaced every 18-72 hours.

[0054] In a feasible embodiment, the decellularizing agent is selected from a mixture of 5-10 U / mL deoxyribonuclease and 50-100 ug / mL ribonuclease.

[0055] In one embodiment, the corneal tissue is removed from the sodium chloride solution and then immersed in a solution containing a decellularizing agent. The decellularizing agent is selected from deoxyribonucleases and ribonucleases, which can act on the DNA and RNA of the corneal tissue, respectively, severing their phosphodiester bonds through hydrolysis, thereby degrading these nucleic acid molecules. This removes genetic material from the tissue, reduces immunogenicity, and maintains the biocompatibility of the material tissue.

[0056] Exemplarily, the concentration of deoxyribonuclease is 5 U / mL, 5.5 U / mL, 6 U / mL, 6.5 U / mL, 7 U / mL, 7.5 U / mL, 8 U / mL, 8.5 U / mL, 9 U / mL, 9.5 U / mL, 10 U / mL, etc. The concentration of ribonuclease is 50 ug / mL, 55 ug / mL, 60 ug / mL, 65 ug / mL, 70 ug / mL, 75 ug / mL, 80 ug / mL, 85 ug / mL, 90 ug / mL, 95 ug / mL, 100 ug / mL, etc.

[0057] If the concentration of deoxyribonuclease is too high, it may increase the risk of nonspecific degradation, adversely affect the matrix lens and its biomechanics, and increase costs. If the concentration of deoxyribonuclease is too low, it may result in incomplete decellularization, affecting the subsequent lens effect. If the concentration of ribonuclease is too high, it may increase the risk of nonspecific degradation, adversely affect the matrix lens and its biomechanics, and affect the subsequent use function of the corneal matrix lens. If the concentration of ribonuclease is too low, it may result in incomplete decellularization, affecting the subsequent lens effect. Therefore, the embodiment of the present application determines that the mixed enzyme solution includes: 5-10U / mL deoxyribonuclease and 50-100ug / mL ribonuclease.

[0058] In one feasible embodiment, the duration of the decellularization treatment is 24 to 72 hours; for example, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 35 hours, 40 hours, 45 hours, 50 hours, 55 hours, 60 hours, 65 hours, 70 hours, 71 hours, 72 hours, etc.

[0059] Step S30: placing the decellularized corneal tissue in a voriconazole solution so that the voriconazole is loaded on the decellularized corneal tissue to obtain an antifungal corneal stromal lens.

[0060] In one embodiment, a voriconazole solution is prepared, wherein the concentration of voriconazole in the voriconazole solution is 0.5 to 2 mg / mL; for example, 0.5 mg / mL, 0.6 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.4 mg / mL, 1.6 mg / mL, 1.8 mg / mL, 2 mg / mL, etc. The decellularized corneal tissue is placed in the voriconazole solution for 4 to 8 hours; for example, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, etc., to achieve voriconazole loading and produce an antifungal corneal stromal lens. Voriconazole can inhibit the activity of key enzymes in fungal cell membranes, which play a key role in the biosynthesis of ergosterol in fungal cell membranes. Therefore, by inhibiting the activity of this enzyme, voriconazole can block the synthesis pathway of ergosterol, which is an important component of the fungal cell membrane. The obstruction of its synthesis will lead to the loss of cell membrane structure and function, thereby destroying the integrity of the fungal cells and ultimately leading to the death of the fungus, achieving an antifungal effect. The drug-loaded corneal stromal lens used for infected lesions can improve the bioavailability and antibacterial effect of ocular drugs for fungal keratitis.

[0061] In this embodiment, the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens extraction (SMILE procedure) is used as a tissue material, which retains the normal tissue structure of the cornea. The obtained corneal tissue is cleaned and disinfected, and then decellularized by a decellularizing agent to remove the cellular components of the corneal tissue and reduce immunogenicity; the decellularized corneal tissue does not produce significant changes in biochemical properties such as tissue expansion rate, in vitro degradation rate, biomechanics, and light transmittance, and the tissue structure of the corneal stromal lens is retained to the maximum extent. The cell voids generated after decellularization can load more drugs to achieve drug loading. At the same time, the decellularized corneal tissue can maintain good stability and functionality after implantation into the eye as a corneal material, and therefore can be used as an ideal drug sustained-release carrier to improve the bioavailability of the drug. Further, the decellularized corneal tissue is placed in a voriconazole solution to achieve the loading of voriconazole in the corneal tissue to produce an antifungal corneal stromal lens. Because corneal tissue itself has good light transmittance, the resulting antifungal corneal stromal lens does not significantly affect the patient's vision after implantation, ensuring the patient's visual quality and quality of life. Voriconazole can inhibit the activity of key enzymes in fungal cell membranes, which play a key role in the biosynthesis of ergosterol in fungal cell membranes. Therefore, by inhibiting the activity of this enzyme, voriconazole can block the synthesis pathway of ergosterol, an important component of fungal cell membranes. Blocked ergosterol synthesis leads to loss of cell membrane structure and function, further damaging the integrity of fungal cells and ultimately causing fungal death, achieving an antifungal effect. The corneal stromal lens loaded with voriconazole can be used as a drug sustained-release system to improve the bioavailability of ocular drugs for fungal keratitis.

[0062] In order to make the details and operations of the above embodiments of the present application clearly understood by those skilled in the art, and to significantly demonstrate the improved performance of the embodiments of the present application, the above technical solutions are illustrated by multiple embodiments below.

[0063] Example 1

[0064] (1) Obtain corneal tissue (central thickness of 100 μm) removed by femtosecond laser small incision corneal lenticule extraction;

[0065] (2) Rinse the corneal tissue with PBS three times, 15 minutes each time;

[0066] (3) Soak the corneal tissue in a mixed solution containing 0.05 mg / mL penicillin and 0.1 mg / mL streptomycin for 2 h, and then rinse with PBS;

[0067] (4) The cleaned and disinfected corneal tissue was immersed in a 1.5 M sodium chloride solution for 30 h, taken out, immersed in a mixed enzyme solution for 48 h, and then rinsed with PBS to obtain acellular corneal tissue, wherein the mixed enzyme solution includes: 5 U / mL deoxyribonuclease and 50 μg / mL ribonuclease.

[0068] Comparative Example 1

[0069] (1) Obtain corneal tissue (central thickness of 100 μm) removed by femtosecond laser small incision corneal lenticule extraction.

[0070] HE staining (Hematoxylin-Eosin staining) and DAPI staining (4',6-diamidino-2-phenylindole) were performed on Example 1 and Comparative Example 1, respectively. Figure 2 ,in, Figure 2 (a) is the HE staining result of Comparative Example 1, Figure 2 (b) is the DAPI staining result of Comparative Example 1, Figure 2 (c) is the HE staining result of Example 1, Figure 2 (d) is the DAPI staining result of Example 1. Figure 2 (c) and Figure 2 In (d), no residual cellular components were found in the corneal tissue, while the collagen structure was neatly arranged and relatively intact, indicating that the decellularization treatment was effective.

[0071] The light transmittance test was carried out on Example 1 and Comparative Example 1, and the results were as follows: Figure 3 .Depend on Figure 3 It can be seen that the transmittance of Example 1 at 555 nm is greater than 90%, which is similar to that of Comparative Example 1, and the transmittance stability is good.

[0072] In vitro degradation tests were performed on Example 1 and Comparative Example 1, and the results were shown in Table 1. Figure 4 .Depend on Figure 4 It can be seen that the in vitro degradation time of Example 1 is 108-120 hours, which is similar to that of Comparative Example 1 (96-108 hours). In summary, the decellularization treatment does not significantly affect the light transmittance and in vitro degradation of the corneal tissue.

[0073] The transmission electron microscope test was performed on Example 1 and Comparative Example 1, and the results were shown in Table 1. Figure 5 ,in, Figure 5 (a) is a schematic diagram of the transmission electron microscope test results of Comparative Example 1, Figure 5 (b) is a schematic diagram of the transmission electron microscope test results of Example 1; Figure 5It can be seen that the collagen structure of the corneal tissue after decellularization is neatly arranged and relatively complete, and no stromal cells are observed.

[0074] Biomechanical tests were performed on Example 1 and Comparative Example 1. The results are shown in Table 1:

[0075] Table 1:

[0076]

[0077] According to the experimental results in Table 1, decellularization does not have a significant impact on the biomechanics of the cornea.

[0078] Example 2

[0079] (1) The acellular corneal tissue prepared in Example 1 was placed in a 0.5 mg / mL voriconazole solution and soaked at room temperature for 4 h;

[0080] (2) Take it out and quickly wash away the drugs attached to the surface with PBS to prepare the antifungal corneal stromal lens.

[0081] Example 3

[0082] (1) The acellular corneal tissue prepared in Example 1 was placed in a voriconazole solution with a concentration of 1 mg / mL and soaked at room temperature for 4 h;

[0083] (2) Take it out and quickly wash away the drugs attached to the surface with PBS to prepare the antifungal corneal stromal lens.

[0084] Example 4

[0085] (1) The acellular corneal tissue prepared in Example 1 was placed in a 2 mg / mL voriconazole solution and soaked at room temperature for 4 h;

[0086] (2) Take it out and quickly wash away the drugs attached to the surface with PBS to prepare the antifungal corneal stromal lens.

[0087] The present application determined that the minimum inhibitory concentration of voriconazole against fungi is 0.06-8 mg / L. Examples 2 to 4 were placed in 1 mL of PBS solution release medium and released in a 37°C constant temperature shaker (speed: 50 rpm). The release medium was completely removed after 1 hour, 2 hours, 4 hours, 8 hours, and 16 hours, and the released voriconazole concentration and the cumulative drug release at each time were measured. Fresh PBS solution was then replaced. The results are shown in Table 2 below.

[0088] Table 2:

[0089]

[0090] According to the results in Table 2, the lenses prepared after decellularization can achieve drug loading and sustained release, and the concentration of released voriconazole can reach the inhibitory concentration, thereby achieving a better antifungal effect and improving the bioavailability of the drug.

[0091] The above are only preferred embodiments of the present application and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of the present invention.

Claims

1. A method for preparing an antifungal corneal stromal lens, characterized in that: The method comprises the following steps: Obtaining corneal tissue removed by femtosecond laser small incision corneal stromal lens removal, wherein the central thickness of the corneal tissue is 80 to 150 μm; Cleaning and disinfecting the corneal tissue, and performing decellularization treatment with a decellularizing agent to obtain decellularized corneal tissue; The decellularized cornea tissue is placed in a voriconazole solution so that the voriconazole is loaded on the decellularized cornea tissue, thereby obtaining an antifungal corneal stroma lens.

2. The method according to claim 1, characterized in that The concentration of voriconazole in the voriconazole solution is 0.5-2 mg / mL.

3. The method according to claim 1, characterized in that The soaking time in the voriconazole solution is 4 to 8 hours.

4. The method according to claim 1, characterized in that Before the step of decellularizing the corneal tissue using a decellularizing agent, the method further comprises: The corneal tissue is immersed in a 1.5-2M sodium chloride solution for 24-48 hours.

5. The method according to claim 1, characterized in that The decellularizing agent is selected from: a mixed enzyme of 5-10 U / mL deoxyribonuclease and 50-100 ug / mL ribonuclease.

6. The method according to claim 1, characterized in that The duration of the decellularization treatment is 24 to 72 hours.

7. The method according to claim 1, characterized in that The step of cleaning and disinfecting the corneal tissue comprises: Washing the corneal tissue with a buffer solution; Sterilize by soaking in a mixed solution of penicillin and streptomycin.

8. The method according to claim 7, characterized in that The washing times of the buffer solution are 3 to 4 times, and each washing time is 15 to 20 minutes.

9. The method according to claim 7, characterized in that The duration of the immersion disinfection is 1 to 5 hours.

10. An antifungal corneal stromal lens, characterized in that: The antifungal corneal stromal lens is prepared by the method according to any one of claims 1 to 9.

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