Cornea stroma lens for inhibiting multi-drug-resistant bacteria and preparation method of cornea stroma lens

Abstract

The invention discloses a cornea stroma lens capable of inhibiting multiple drug-resistant bacteria and a preparation method of the cornea stroma lens, relates to the technical field of tissue engineering, and discloses the preparation method of the cornea stroma lens capable of inhibiting the multiple drug-resistant bacteria, which comprises the following steps: providing a cornea stroma lens raw material, the cornea stroma lens raw material is cornea tissue taken out through femtosecond laser small incision cornea stroma lens extraction; carrying out decellularization treatment on the cornea stroma lens raw material to obtain a decellularized cornea stroma lens; performing cross-linking treatment on the decellularized cornea stroma lens to obtain a decellularized cross-linked cornea stroma lens; and placing the decellularized cross-linked cornea stroma lens in a nano-silver solution to carry out nano-silver loading so as to prepare the cornea stroma lens for inhibiting multiple drug-resistant bacteria. According to the cornea stroma lens for inhibiting the multi-drug-resistant bacteria, the bioavailability of eye medicine is improved, and the problem of drug resistance is solved.

Description

Technical Field

[0001] The present application relates to the field of tissue engineering technology, and in particular to a corneal stromal lens for inhibiting multi-drug resistant bacteria and a preparation method thereof. Background Art

[0002] As an ophthalmic disease with a high incidence rate and prone to blindness, infectious corneal ulcers face severe challenges in their treatment. At present, the main treatment method relies on topical eye drops. Although this method of administration is direct, it is limited by the special physiological structure of the eye and frequent physiological activities, such as blinking and tearing, which makes the retention of drugs on the surface of the eyeball and conjunctiva extremely limited. In addition, due to the numerous barrier functions of the eye, the amount of drugs that can actually penetrate into the eye and exert a therapeutic effect is less than 5% of the total amount, which greatly reduces the bioavailability of the drug and affects the treatment effect. In addition, with the widespread use of antimicrobial drugs, pathogens have gradually developed drug resistance, which has greatly reduced the effectiveness of traditional therapeutic drugs.

[0003] The above contents are only used to assist in understanding the technical solution of the present application and do not constitute an admission that the above contents are prior art. Summary of the invention

[0004] The main purpose of the present application is to provide a corneal stromal lens for inhibiting multidrug-resistant bacteria and a preparation method thereof, aiming to provide a corneal stromal lens for inhibiting multidrug-resistant bacteria to improve the bioavailability of ocular drugs and overcome the problem of drug resistance.

[0005] To achieve the above-mentioned purpose, the present invention provides a method for preparing a corneal stromal lens for inhibiting multi-drug resistant bacteria, the method comprising:

[0006] Providing corneal stromal lens raw material, wherein the corneal stromal lens raw material is corneal tissue removed by femtosecond laser small incision corneal stromal lens removal surgery;

[0007] Decellularizing the corneal stromal lens raw material to obtain a decellularized corneal stromal lens;

[0008] Cross-linking the acellular corneal stromal lens to obtain a acellular cross-linked corneal stromal lens;

[0009] The decellularized cross-linked corneal stroma lens is placed in a nanosilver solution to load the nanosilver, thereby preparing a corneal stroma lens capable of inhibiting multi-drug resistant bacteria.

[0010] In one embodiment, the concentration of nanosilver in the nanosilver solution is 500-1500 ug / mL.

[0011] In one embodiment, the immersion time in the nanosilver solution is 2 to 8 hours.

[0012] In one embodiment, the step of performing acellular treatment on the corneal stromal lens raw material to obtain a decellularized corneal stromal lens comprises:

[0013] The corneal stromal lens raw material is sequentially placed in a sodium chloride solution and a mixed enzyme solution for immersion to obtain the decellularized corneal stromal lens, wherein the mixed enzyme solution comprises: deoxyribonuclease and ribonuclease.

[0014] In one embodiment, the mixed enzyme solution includes: 5-10 U / mL deoxyribonuclease and 50-100 ug / mL ribonuclease;

[0015] And / or, the soaking time in the mixed enzyme solution is 24 to 72 hours.

[0016] In one embodiment, the molar concentration of the sodium chloride solution is 1.5 to 2 M;

[0017] And / or, the soaking time in the sodium chloride solution is 24 to 48 hours.

[0018] In one embodiment, before the step of performing acellular treatment on the corneal stromal lens raw material, the method further comprises:

[0019] Washing the corneal stromal lens material with a buffer solution, wherein the washing times are 3 to 4 times, and each washing time is 15 to 20 minutes;

[0020] And / or, the corneal stromal lens raw material is immersed and disinfected by a mixed solution of penicillin and streptomycin, wherein the immersion and disinfection time is 1 to 5 hours.

[0021] In one embodiment, the step of cross-linking the acellular corneal stromal lens to obtain the acellular cross-linked corneal stromal lens comprises:

[0022] Providing a crosslinking agent solution, wherein the crosslinking agent solution comprises: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide;

[0023] The decellularized corneal stromal lens is placed in the crosslinking agent solution for immersion to obtain the decellularized crosslinked corneal stromal lens.

[0024] In one embodiment, the molar to mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the acellular corneal stromal lens in the crosslinking agent solution is (0.01-0.25 mmol): 1 mg;

[0025] and / or, the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide in the crosslinking agent solution is (5-15):(1-5);

[0026] And / or, the immersion time in the cross-linking agent solution is 4 to 8 hours.

[0027] In one embodiment, the central thickness of the corneal stromal lens material is 80-150 μm.

[0028] In addition, to achieve the above-mentioned purpose, the embodiments of the present application also provide a corneal stromal lens for inhibiting multi-drug resistant bacteria, which is prepared by the method described above.

[0029] One or more technical solutions proposed in this application have at least the following technical effects: First, the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens removal is used as tissue material; because the femtosecond laser small incision corneal stromal lens removal (SMILE procedure) cuts off less collagen fibers of the corneal stroma and retains more normal tissue structure and morphology of the cornea, it can provide a more complete and biomechanically stable corneal tissue material. Secondly, the cell components of the corneal stromal lens raw material are effectively removed by decellularization, the immunogenicity is reduced, and the rejection rate of the corneal stromal lens is reduced; and the cell gaps generated after decellularization can load more drugs to achieve drug loading. Thirdly, the strength between the fibers of the corneal stromal lens is increased to a certain extent by cross-linking treatment, so that the prepared corneal stromal lens can maintain long stability and functionality in the in vivo environment, become an ideal drug sustained-release carrier, and can improve the bioavailability of the drug; and the corneal stromal lens has good light transmittance, and will not have a significant impact on the patient's vision after implantation in the eye, ensuring the patient's visual quality and quality of life. Finally, the decellularized cross-linked corneal stroma lens was placed in a nanosilver solution to load the lens with nanosilver, thereby producing a corneal stroma lens sustained-release system that inhibits multi-drug resistant bacteria. Since the antibacterial mechanism of nanosilver is different from that of traditional antibiotics, it does not directly target specific metabolic pathways or enzyme systems of microorganisms, but kills bacteria through multiple pathways such as destroying cell walls, cell membranes, and life substances. Therefore, it is difficult for nanosilver to develop drug resistance. Instead, it can penetrate deep into the tissue through its strong permeability to kill drug-resistant bacteria hidden deep inside, thereby effectively avoiding the generation and spread of drug resistance and overcoming the problem of drug resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is a flow chart of a method for preparing a corneal stromal lens for inhibiting multi-drug resistant bacteria according to an embodiment of the present application;

[0031] Figure 2It is a schematic diagram of the results of HE staining and DAPI staining of Example 1 and Comparative Example 1 of the present application;

[0032] Figure 3 Schematic diagram of light transmittance test results of Example 1 and Comparative Example 1 of the present application;

[0033] Figure 4 This is a schematic diagram of the in vitro degradation test results of Example 1 and Comparative Example 1 of the present application;

[0034] Figure 5 This is a schematic diagram of the scanning electron microscope results of Example 2 of the present application;

[0035] Figure 6 It is a schematic diagram of the transmission electron microscopy results of Examples 1 and 2 of the present application and Comparative Example 1;

[0036] Figure 7 Schematic diagram of light transmittance test results of Example 2 and Comparative Example 1 of the present application;

[0037] Figure 8 This is a schematic diagram of the in vitro degradation test results of Example 2 and Comparative Example 1 of the present application;

[0038] Fig. 9 Schematic diagram of the swelling test results of Example 2 and Comparative Example 1 of the present application.

[0039] The realization of the purpose, functional features and advantages of this application will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0041] Hereinafter, the embodiment of the method for recovering lithium from waste lithium iron phosphate batteries of the present application is specifically disclosed with appropriate reference to the accompanying drawings. However, there may be cases where unnecessary detailed descriptions are omitted. For example, there are cases where detailed descriptions of well-known matters and repeated descriptions of actually the same structures are omitted. This is to avoid the following description from becoming unnecessarily lengthy and to facilitate the understanding of those skilled in the art. In addition, the accompanying drawings and the following description are provided for those skilled in the art to fully understand the present application and are not intended to limit the subject matter described in the claims.

[0042] The "range" disclosed in the present application is defined in the form of a lower limit and an upper limit, and a given range is defined by selecting a lower limit and an upper limit, and the selected lower limit and upper limit define the boundaries of the particular range. The range defined in this way can be inclusive or exclusive of the end values, and can be arbitrarily combined, that is, any lower limit can be combined with any upper limit to form a range. For example, if a range of 60 to 120 and 80 to 110 is listed for a particular parameter, it is understood that a range of 60 to 110 and 80 to 120 is also expected. In addition, if the minimum range values ​​1 and 2 are listed, and if the maximum range values ​​3, 4 and 5 are listed, the following ranges can all be expected: 1 to 3, 1 to 4, 1 to 5, 2 to 3, 2 to 4 and 2 to 5. In the present application, unless otherwise specified, the numerical range "a to b" represents an abbreviation of any real number combination between a and b, where a and b are both real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" are listed in this document, and "0-5" is just an abbreviation of these numerical combinations. In addition, when a parameter is expressed as an integer ≥ 2, it 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.

[0043] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0044] 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.

[0045] If there is no special explanation, all steps of the present application can be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), which means 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), which means 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.

[0046] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0047] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": 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).

[0048] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and understandable, the technical solution of the present application is further described below in conjunction with the accompanying drawings and embodiments. However, the present application is not limited to the listed embodiments, and should also include any other known changes within the scope of the rights claimed in the present application.

[0049] The term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present application. The term "in one embodiment" that appears in different places in 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.

[0050] 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.

[0051] In conventional technology, the main treatment for infectious corneal ulcers relies on topical eye drops. Although this method of administration is direct, the bioavailability of the drug is low. The ocular surface drug delivery system can effectively improve the bioavailability of the drug. Conventional ocular surface drug delivery systems mainly include: drug-loaded amniotic membrane and drug-loaded corneal contact lenses; among them, drug-loaded amniotic membrane treats 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. After the drug is loaded, the drug-loaded corneal contact lens is hydrated, and the transmittance, oxygen permeability and biocompatibility decrease, which in turn causes other eye problems. In addition, with the widespread use of antimicrobial drugs, pathogens have gradually developed drug resistance, which has greatly reduced the effectiveness of traditional therapeutic drugs.

[0052] The present application provides a solution. First, the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens removal is used as tissue material; because the femtosecond laser small incision corneal stromal lens removal (SMILE procedure) cuts off less collagen fibers of the corneal stroma and retains more normal tissue structure and morphology of the cornea, it can provide a more complete and biomechanically stable corneal tissue material. Secondly, the cell components of the corneal stromal lens raw material are effectively removed by decellularization, the immunogenicity is reduced, and the rejection rate of the corneal stromal lens is reduced; and the cell gaps produced after decellation can load more drugs to achieve drug loading. Again, the strength between the fibers of the corneal stromal lens is increased to a certain extent by cross-linking treatment, so that the prepared corneal stromal lens can maintain a long stability and functionality in the in vivo environment, become an ideal drug sustained-release carrier, and can improve the bioavailability of the drug; and the corneal stromal lens has good light transmission performance, and will not have a significant impact on the patient's vision after implantation in the eye, ensuring the patient's visual quality and quality of life. Finally, the decellularized cross-linked corneal stroma lens was placed in a nanosilver solution to load the lens with nanosilver, so as to prepare a corneal stroma lens sustained-release carrier that inhibits multi-drug resistant bacteria. Since the antibacterial mechanism of nanosilver is different from that of traditional antibiotics, it does not directly target the specific metabolic pathways or enzyme systems of microorganisms, but kills bacteria through multiple pathways such as destroying cell walls, cell membranes and life substances. Therefore, it is difficult for nanosilver to develop drug resistance. Instead, it can penetrate deep into the tissue through the strong permeability of nanosilver to kill drug-resistant bacteria hidden deep inside, thereby effectively avoiding the generation and spread of drug resistance and overcoming the problem of drug resistance.

[0053] The first aspect of the embodiment of the present application provides a method for preparing a corneal stromal lens for inhibiting multi-drug resistant bacteria, comprising the following steps:

[0054] Step S10, providing corneal stromal lens raw material, wherein the corneal stromal lens raw material is corneal tissue removed by femtosecond laser small incision corneal stromal lens removal surgery;

[0055] In a feasible embodiment, the corneal tissue removed by femtosecond laser small incision corneal stromal lens extraction is used as the corneal stromal lens raw material. Since the femtosecond laser small incision corneal stromal lens extraction (SMILE procedure) cuts less collagen fibers in the corneal stroma and retains more normal tissue structure and morphology of the cornea, it can provide a more complete and biomechanically stable corneal tissue. The corneal tissue can be stored in anhydrous glycerol for subsequent use.

[0056] In one feasible embodiment, the central thickness of the corneal stromal lens material 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 stromal lens material 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, the embodiment of the present application determines that the central thickness of the corneal stromal lens material is 80 to 150 μm.

[0057] Step S20, performing a decellularization process on the corneal stromal lens raw material to obtain a decellularized corneal stromal lens;

[0058] In a feasible embodiment, the corneal stromal lens raw material is subjected to a decellularization treatment to obtain a decellularized corneal stromal lens; the decellularization treatment effectively removes the cellular components of the corneal stromal lens raw material, reduces immunogenicity, and lowers the rejection rate of the corneal stromal lens; and the cell gaps generated after decellularization can be loaded with more drugs to achieve drug loading.

[0059] In one feasible implementation, step S20, performing a decellularization process on the corneal stromal lens raw material to obtain a decellularized corneal stromal lens comprises:

[0060] Step S21, placing the corneal stromal lens raw material into a sodium chloride solution and a mixed enzyme solution in turn for immersion to obtain a cell-free corneal stromal lens, wherein the mixed enzyme solution comprises: deoxyribonuclease and ribonuclease.

[0061] In a feasible embodiment, a sodium chloride solution and a mixed enzyme solution are provided, wherein the mixed enzyme solution includes: deoxyribonuclease and ribonuclease. The corneal stromal lens raw material is soaked in the sodium chloride solution; the high osmotic pressure characteristics of the sodium chloride solution are used to make the water in the cells penetrate to the outside of the cells, causing the cells to dehydrate and rupture, which is conducive to removing the cell 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 corneal stromal lens is taken out of the sodium chloride solution and then soaked in the mixed enzyme solution. The mixed enzyme solution includes deoxyribonuclease and ribonuclease, which can act on the DNA and RNA of the corneal stromal lens respectively, cut their phosphodiester bonds through hydrolysis, thereby degrading these nucleic acid molecules, and removing the genetic material in the tissue, reducing immunogenicity, and improving the biocompatibility of the material. In a feasible embodiment, 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 sodium chloride solution is too high, it may cause changes in tissue structure and loss of function; while a sodium chloride solution with too low a concentration may also lead to poor decellularization effect. Therefore, the molar concentration of the sodium chloride solution is determined to be 1.5-2M in the present embodiment.

[0062] In one feasible embodiment, the immersion 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.

[0063] In one feasible embodiment, the mixed enzyme solution includes: 5-10U / mL deoxyribonuclease and 50-100Ug / mL ribonuclease; for example, the concentration of deoxyribonuclease is 5U / mL, 5.5U / mL, 6U / mL, 6.5U / mL, 7U / mL, 7.5U / mL, 8U / mL, 8.5U / mL, 9U / mL, 9.5U / mL, 10U / mL, etc. The concentration of ribonuclease is 50ug / mL, 55ug / mL, 60ug / mL, 65ug / mL, 70ug / mL, 75ug / mL, 80ug / mL, 85ug / mL, 90ug / mL, 95ug / mL, 100ug / mL, etc.

[0064] If the concentration of deoxyribonuclease is too high, the risk of nonspecific degradation may increase, adversely affecting the matrix lens and its biomechanics, and increasing costs. If the concentration of deoxyribonuclease is too low, the decellularization process may not be thorough, affecting the subsequent lens effect. If the concentration of ribonuclease is too high, the risk of nonspecific degradation may increase, adversely affecting the matrix lens and its biomechanics, affecting the subsequent use function of the corneal matrix lens. If the concentration of ribonuclease is too low, the decellularization process may not be thorough, 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.

[0065] In one feasible embodiment, the soaking time in the mixed enzyme solution 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.

[0066] In a feasible implementation manner, before the step of decellularizing the corneal stromal lens raw material to obtain the decellularized corneal stromal lens in step S20, the method further includes:

[0067] The corneal stromal lens material is obtained, and washed with a buffer solution for 3 to 4 times, each time for 15 to 20 minutes; it is 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.

[0068] In a feasible implementation, after the step of soaking the corneal stromal lens raw material in sodium chloride solution and mixed enzyme solution in turn in step S21, the method further includes: rinsing with buffer solution for 48 to 96 hours, wherein the buffer solution is uniformly oscillated on a shaker at 100 r / h and the buffer solution is replaced every 18 to 72 hours.

[0069] Step S30, cross-linking the decellularized corneal stromal lens to obtain a decellularized cross-linked corneal stromal lens;

[0070] In a feasible embodiment, the decellularized corneal stromal lens is cross-linked to obtain a decellularized cross-linked corneal stromal lens. The cross-linking treatment increases the strength between the fibers of the corneal stromal lens to a certain extent, making the corneal stromal lens more resistant to the effects of various degradative enzymes in the eye, reducing the degradation rate of collagen, and enabling the prepared corneal stromal lens to maintain long-term stability and functionality in the in vivo environment, becoming an ideal drug sustained-release carrier, and improving the bioavailability of drugs; and the corneal stromal lens itself has good light transmittance, so it will not have a significant impact on the patient's vision after being implanted in the eye, ensuring the patient's visual quality and quality of life.

[0071] In one feasible implementation, step S30, cross-linking the decellularized corneal stromal lens to obtain the decellularized cross-linked corneal stromal lens comprises:

[0072] Step S31, providing a crosslinking agent solution, wherein the crosslinking agent solution includes: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide;

[0073] In a feasible embodiment, a crosslinking agent solution is prepared, wherein the crosslinking agent in the crosslinking agent solution is selected from: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide.

[0074] In a feasible embodiment, the molar to mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the acellular corneal stromal lens in the cross-linking agent solution is (0.01-0.25mmol):1mg; for example, the molar to mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the acellular corneal stromal lens is 0.01mmol:1mg, 0.02mmol:1mg, 0.03mmol:1mg, 0.04mmol:1mg, 0.06mmol:1mg, 0.08mmol:1mg, 0.1mmol:1mg, 0.12mmol:1mg, 0.14mmol:1mg, 0.16mmol:1mg, 0.18mmol:1mg, 0.2mmol:1mg, 0.21mmol:1mg, 0.22mmol:1mg, 0.23mmol:1mg, 0.24mmol:1mg, 0.25mmol:1mg, etc.

[0075] In one feasible embodiment, the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide in the cross-linking agent solution is (5-15):(1-5); for example, the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide in the cross-linking agent solution is 5:1, 6:1.5, 7:2, 8:2.5, 9:3, 10:3.5, 11:4, 12:4.5, 13:5, 14:1, 15:1.5, etc.

[0076] Through cross-linking, the connection between the collagen molecules of the corneal stroma lens is made tighter, and the original gaps are filled with new covalent bonds, thereby reducing the gaps between the fibers, allowing more drug molecules to be loaded per unit volume, thereby improving the drug loading efficiency; at the same time, the cross-linked collagen network structure can better protect the drug molecules and prevent them from being degraded or inactivated during the loading and release process, thereby ensuring the stability and effectiveness of the drug, and then improving the bioavailability of the drug after loading.

[0077] Step S32, placing the decellularized corneal stromal lens into a crosslinking agent solution for immersion to obtain a decellularized crosslinked corneal stromal lens.

[0078] In a feasible embodiment, the decellularized corneal stromal lens is immersed in a crosslinking agent solution for 4 to 8 hours, taken out and rinsed with a buffer solution for 2 to 5 times, each time for 10 to 20 minutes, to obtain a decellularized crosslinked corneal stromal lens.

[0079] Illustratively, the soaking time may be 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, etc.

[0080] Step S40, placing the decellularized cross-linked corneal stromal lens in a nanosilver solution to load the nanosilver, thereby obtaining a corneal stromal lens capable of inhibiting multidrug-resistant bacteria.

[0081] In one feasible embodiment, a nanosilver solution is prepared, wherein the concentration of nanosilver in the nanosilver solution is 500-1500ug / mL; for example, 500ug / mL, 600ug / mL, 700ug / mL, 800ug / mL, 900ug / mL, 1000ug / mL, 1100ug / mL, 1200ug / mL, 1300ug / mL, 1400ug / mL, 1500ug / mL, etc. The decellularized cross-linked corneal stromal lens is placed in the nanosilver solution for 2-8h; for example, 2h, 3h, 4h, 5h, 6h, 7h, 8h, etc., to achieve the loading of nanosilver and obtain a drug-resistant corneal stromal lens. Since the antibacterial mechanism of nanosilver is different from that of traditional antibiotics, it does not directly target the specific metabolic pathways or enzyme systems of microorganisms, but kills bacteria through multiple pathways such as destroying cell walls, cell membranes and vital substances. Therefore, it is difficult for nanosilver to be overcome by microorganisms that develop drug resistance. On the contrary, nanosilver can use its powerful permeability to penetrate deep into tissues and kill drug-resistant bacteria hidden deep inside, thereby effectively avoiding the generation and spread of drug resistance and overcoming the problem of drug resistance.

[0082] In this embodiment, firstly, the corneal stromal lens removed by femtosecond laser small incision corneal stromal lens extraction is used as tissue material; since femtosecond laser small incision corneal stromal lens extraction (SMILE procedure) cuts less collagen fibers in the corneal stroma and retains more normal tissue structure and morphology of the cornea, it can provide a more complete and biomechanically stable corneal tissue. Secondly, the cell components of the corneal stromal lens raw material are effectively removed by decellularization, the immunogenicity is reduced, and the rejection rate of the corneal stromal lens is reduced; and the cell gaps generated after decellularization can load more drugs to achieve drug loading. Thirdly, the cross-linking treatment can increase the strength between the fibers of the corneal stromal lens to a certain extent, making the corneal stromal lens more resistant to the effects of various degradative enzymes, reducing the degradation rate of collagen, and not having a significant impact on the light transmittance of the cornea itself; the prepared corneal stromal lens can maintain long-term stability and functionality in the in vivo environment, becoming an ideal drug sustained-release carrier, which can improve the bioavailability of drugs; and the same light transmittance as the cornea means that the corneal stromal lens will not have a significant impact on the patient's vision after being implanted in the eye, ensuring the patient's visual quality and quality of life. Finally, the decellularized cross-linked corneal stromal lens is placed in a nanosilver solution to achieve the loading of nanosilver in the lens, so as to prepare a corneal stromal lens sustained-release system that inhibits multi-drug resistant bacteria. Since the antibacterial mechanism of nanosilver is different from that of traditional antibiotics, it does not directly target the specific metabolic pathways or enzyme systems of microorganisms, but kills bacteria through multiple pathways such as destroying cell walls, cell membranes and vital substances. Therefore, it is difficult for nanosilver to develop drug resistance. On the contrary, it can use its powerful permeability to penetrate deep into tissues to kill resistant bacteria hidden deep inside, thereby effectively avoiding the generation and spread of drug resistance and overcoming the problem of drug resistance.

[0083] In order to enable the details and operations of the above embodiments of the present application to be 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 means of multiple embodiments below.

[0084] Example 1

[0085] (1) Obtain corneal tissue (central thickness of 100 μm) removed by femtosecond laser small incision corneal stromal lens extraction to serve as corneal stromal lens raw material;

[0086] (2) Rinse the corneal stromal lens material three times with PBS, each time for 15 min;

[0087] (3) Soaking the corneal stromal lens material in a mixed solution containing 0.05 mg / mL penicillin and 0.1 mg / mL streptomycin for 2 h, and then rinsing with PBS;

[0088] (4) Soaking the cleaned and disinfected corneal stromal lens raw material in a 1.5M sodium chloride solution for 30 hours, taking it out, soaking it in a mixed enzyme solution for 48 hours, and then rinsing it with PBS to obtain a decellularized corneal stromal lens, wherein the mixed enzyme solution includes: 5U / mL deoxyribonuclease and 50ug / mL ribonuclease.

[0089] Comparative Example 1

[0090] (1) Obtain corneal tissue (central thickness of 100 μm) removed by femtosecond laser small incision corneal stromal lens extraction to serve as corneal stromal lens raw material.

[0091] HE staining (Hematoxylin-Eosin staining) and DAPI staining (4',6-diamidino-2-phenylindole) were performed on Example 1 and Comparative Example 1, respectively. The results are shown in Table 1. 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 stroma, while the collagen structure was neatly arranged and relatively intact, indicating that the decellularization treatment was effective.

[0092] The light transmittance of Example 1 and Comparative Example 1 was tested, 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 better.

[0093] In vitro degradation tests were performed on Example 1 and Comparative Example 1. The results are shown in Table 1. Figure 4 .Depend on Figure 4 It can be seen that the in vitro degradation time of Example 1 is 108-120h, which is similar to that of Comparative Example 1 (96-108h). In summary, the decellularization treatment does not affect the light transmittance and in vitro degradation of the corneal tissue too much.

[0094] Example 2

[0095] (1) drying the acellular corneal stromal lens prepared according to Example 1;

[0096] (2) preparing a crosslinking agent solution, wherein the molar to mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the acellular corneal stromal lens in the crosslinking agent solution is 0.01 mmol:1 mg, and the mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysulfosuccinimide is 5:1;

[0097] (3) The dried decellularized corneal stromal lens was immersed in a crosslinking agent solution for 4 hours, and then rinsed with PBS for 3 times, each time for 15 minutes, to obtain a decellularized crosslinked corneal stromal lens.

[0098] The acellular cross-linked corneal stromal lens prepared in Example 2 was tested by scanning electron microscopy. The results are as follows: Figure 5 As shown, Figure 5 (a) is a cross-section of acellular cross-linked corneal stromal lens at multiple locations. Figure 5 (b) is the surface of the decellularized cross-linked corneal stromal lens at multiple locations. Figure 5 It can be seen that filamentous collagen fibers can be seen in the cross section of the acellular cross-linked corneal stromal lens, while the collagen fibers can be seen in a mesh-like configuration on the surface.

[0099] The lenses prepared in Examples 1 and 2 and Comparative Example 1 were tested by transmission electron microscopy. The results are as follows: Figure 6 As shown, Figure 6 (a) is a transmission electron microscope image of the lens prepared in Comparative Example 1, Figure 6 (b) is a transmission electron microscope image of the lens obtained in Example 1, Figure 6 (c) is a transmission electron microscope image of the lens obtained in Example 2. Figure 6 It can be seen that the collagen fibers of the lens obtained after decellularization and cross-linking treatment (Example 2) are arranged more densely.

[0100] The light transmittance of Example 2 and Comparative Example 1 was tested, and the results were as follows: Figure 7 .Depend on Figure 7 It can be seen that the transmittance of Example 2 at 555 nm is greater than 90%, which is similar to that of Comparative Example 1, and the transmittance stability is good. In summary, the cross-linking treatment does not affect the transmittance and in vitro degradation of the lens too much.

[0101] It should be understood that based on Comparative Example 1, a plurality of corneal stromal lens raw materials were obtained, and Figure 3 and Figure 7 ,as well as Figure 4 and Figure 8 Different corneal stromal lens materials obtained in Comparative Example 1 were used respectively. Figure 3 and Figure 7 There is a slight difference in the transmittance of Comparative Example 1, and Figure 4and Figure 8 There are slight differences in the in vitro degradation data of Comparative Example 1.

[0102] The swelling degree test was carried out on Example 2 and Comparative Example 1. The test results are as follows: Fig. 9 shown; according to Fig. 9 The results show that the swelling degree of the lens after decellularized cross-linking treatment is reduced, thereby effectively avoiding the occurrence of edema after implantation into the eye.

[0103] In vitro degradation tests were performed on Example 2 and Comparative Example 1. The results are shown in Table 1. Figure 8 .Depend on Figure 8 It can be seen that the in vitro degradation time of Example 2 is 96 to 108 h, which is similar to that of Comparative Example 1 (96-108 h).

[0104] Biomechanical tests were performed on Examples 1, 2 and Comparative Example 1. The results are shown in Table 1:

[0105] Table 1:

[0106]

[0107] According to the experimental results in Table 1, decellularization will not have a significant impact on the biomechanics of the cornea, while cross-linking can improve the biomechanics of the cornea to a certain extent and increase the strength between the fibers of the corneal stroma lens.

[0108] Example 3

[0109] (1) placing the acellular cross-linked corneal stromal lens prepared in Example 1 in a nanosilver solution with a concentration of 1000 ug / mL and soaking it at room temperature for 4 hours;

[0110] (2) taking it out and quickly washing away the drugs attached to the surface with double distilled water to produce a corneal stromal lens that inhibits multidrug-resistant bacteria.

[0111] Comparative Example 2

[0112] (1) placing the acellular corneal stromal lens prepared in Example 1 in a 1000 ug / mL nanosilver solution and soaking it at room temperature for 4 h;

[0113] (2) Take it out and quickly rinse off the drugs attached to the surface with double distilled water to prepare a cell-free drug-loaded corneal stromal lens.

[0114] The diameter of the nanosilver used in Example 3 and Comparative Example 2 of the present application is 3 nm. The microbroth dilution method is used to detect the growth of 3 nm nanosilver in MH agar medium, and it is determined that the minimum inhibitory concentration of nanosilver against Staphylococcus aureus is 2 ppm, and the minimum inhibitory concentration against Pseudomonas aeruginosa is 0.4 ppm.

[0115] The decellular drug-loaded corneal stromal lenses of Example 3 and Comparative Example 2 were placed in 1 mL of double-distilled aqueous solution release medium, and the drug was released in a constant temperature shaker box at 37°C (rotation speed: 50 r / min); the release medium was completely taken out at 0.5h, 1h, 2h, 3h, and 4h, respectively, and the released nanosilver concentration and the cumulative drug release amount each time were measured, and a new double-distilled aqueous solution was replaced at the same time. The results are shown in Table 2 below.

[0116] Table 2:

[0117]

[0118] According to the results in Table 2, the lens prepared by decellularization (Comparative Example 2) can achieve drug loading, and the concentration of nanosilver released after loading can reach the antibacterial concentration. The lens prepared by further cross-linking (Example 3) can further increase the drug release of the lens and reach the in vitro antibacterial concentration to inhibit multi-drug resistant bacteria and overcome the drug resistance problem.

[0119] The above are only preferred embodiments of the present application, and do not limit the scope of the present application. For those skilled in the art, the present application may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the scope of patent protection of the present application.

Claims

1. A method for preparing a corneal stromal lens for inhibiting multi-drug resistant bacteria, characterized in that: The method comprises the following steps: Providing corneal stromal lens raw material, wherein the corneal stromal lens raw material is corneal tissue removed by femtosecond laser small incision corneal stromal lens removal surgery; Decellularizing the corneal stromal lens raw material to obtain a decellularized corneal stromal lens; Cross-linking the acellular corneal stromal lens to obtain a acellular cross-linked corneal stromal lens; The decellularized cross-linked corneal stroma lens is placed in a nanosilver solution to load the nanosilver, thereby preparing a corneal stroma lens capable of inhibiting multi-drug resistant bacteria.

2. The method according to claim 1, characterized in that The concentration of nanosilver in the nanosilver solution is 500-1500 ug / mL.

3. The method according to claim 1, characterized in that The immersion time in the nanosilver solution is 2 to 8 hours.

4. The method according to claim 1, characterized in that The step of performing acellular treatment on the corneal stromal lens raw material to obtain a decellularized corneal stromal lens comprises: The corneal stromal lens raw material is sequentially placed in a sodium chloride solution and a mixed enzyme solution for immersion to obtain the decellularized corneal stromal lens, wherein the mixed enzyme solution comprises: deoxyribonuclease and ribonuclease.

5. The method according to claim 4, characterized in that The mixed enzyme solution comprises: 5-10 U / mL deoxyribonuclease and 50-100 ug / mL ribonuclease; And / or, the soaking time in the mixed enzyme solution is 24 to 72 hours.

6. The method according to claim 4, characterized in that The molar concentration of the sodium chloride solution is 1.5 to 2 M; And / or, the soaking time in the sodium chloride solution is 24 to 48 hours.

7. The method according to claim 1, characterized in that The step of cross-linking the decellularized corneal stromal lens to obtain the decellularized cross-linked corneal stromal lens comprises: Providing a crosslinking agent solution, wherein the crosslinking agent solution comprises: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide; The decellularized corneal stromal lens is placed in the crosslinking agent solution for immersion to obtain the decellularized crosslinked corneal stromal lens.

8. The method according to claim 7, characterized in that The molar to mass ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to the acellular corneal stromal lens in the crosslinking agent solution is (0.01-0.25 mmol): 1 mg; and / or, the mass ratio between 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysulfosuccinimide in the crosslinking agent solution is (5-15):(1-5); And / or, the immersion time in the cross-linking agent solution is 4 to 8 hours.

9. The method according to claim 1, characterized in that The central thickness of the corneal stromal lens material is 80 to 150 μm.

10. A corneal stromal lens for inhibiting multi-drug resistant bacteria, characterized in that: The corneal stromal lens for inhibiting multi-drug resistant bacteria is prepared by the method according to any one of claims 1 to 9.

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