Eutectoid gel as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biomedical materials, in particular to eutectoid gel as well as a preparation method and application thereof. The eutectoid gel comprises a deep eutectic solvent and lysozyme fibers, the effective components of the deep eutectic solvent comprise choline chloride, gallic acid and urea. The eutectoid gel has high DPPH free radical scavenging capacity and ABTS free radical scavenging capacity (anti-inflammatory performance), can effectively inhibit growth (antibacterial performance) of escherichia coli, staphylococcus aureus and pseudomonas aeruginosa, further has a good permeation effect (cornea permeability) on cornea, and can be applied to treatment of bacterial keratitis, such as keratitis, keratitis and the like. The invention provides a new theoretical basis and experimental basis for the development of a new medicine for clinically treating bacterial keratitis, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and in particular to a eutectoid gel and a preparation method and application thereof. Background Art

[0002] Bacterial keratitis is an ophthalmic disease caused by bacterial infection. Its main pathogens include Pseudomonas aeruginosa and Staphylococcus aureus. The clinical manifestations of the disease include corneal congestion, edema, exudation and decreased vision. In severe cases, it can lead to corneal ulcers, perforations and even vision loss. In order to treat bacterial keratitis, antibiotics are often used clinically. Common antibiotics include fluoroquinolones, aminoglycosides and vancomycin. However, long-term or excessive use of these antibiotics will lead to increased bacterial resistance, thereby reducing the treatment effect and making the treatment of bacterial keratitis more difficult. There is an urgent need to develop new strategies for the treatment of bacterial keratitis.

[0003] Deep eutectic solvents (DES) are eutectic mixtures composed of hydrogen bond donors and acceptors. They offer advantages such as ease of preparation, high solubility, good biocompatibility, and robust tunability, showing broad application prospects in biomedical materials, cosmetics, and flexible devices. While DESs have made significant progress in various applications, research on their application in the treatment of ophthalmic diseases, particularly bacterial keratitis, remains scarce. Summary of the Invention

[0004] The present invention provides a eutectic gel comprising a deep eutectic solvent and lysozyme fibers; the eutectic gel has excellent anti-inflammatory properties, antibacterial properties and corneal permeability, and can be used to treat bacterial keratitis.

[0005] The present invention also provides a method for preparing the above-mentioned eutectoid gel, which can produce an eutectoid gel with excellent anti-inflammatory, antibacterial and corneal permeability. The method is simple, low-cost and suitable for large-scale production.

[0006] The present invention also provides a use of the aforementioned eutectoid gel in the preparation of a drug for treating bacterial keratitis. Research by the inventors has shown that the eutectoid gel provided by the present invention has excellent free radical scavenging ability, inhibiting the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. It also has a good corneal permeability effect. Therefore, the eutectoid gel can be used to prepare a drug for treating bacterial keratitis.

[0007] The present invention also provides an antibacterial agent comprising the aforementioned eutectoid gel; the antibacterial agent can inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Research by the present inventors has shown that the eutectoid gel provided by the present invention can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, and therefore can be used to prepare an antibacterial agent.

[0008] A first aspect of the present invention provides a eutectic gel comprising a deep eutectic solvent and lysozyme fibers;

[0009] The effective components of the deep eutectic solvent consist of choline chloride, gallic acid and urea.

[0010] In the eutectoid gel described above, in the deep eutectic solvent, the mass percentage of choline chloride is 75%-85%, the mass percentage of gallic acid is 5%-15%, the mass percentage of urea is 5%-15%, and the sum of the active ingredients is 100%.

[0011] In the eutectoid gel described above, in the deep eutectic solvent, the mass percentage of choline chloride is 80%, the mass percentage of gallic acid is 10%-15%, the mass percentage of urea is 5%-10%, and the sum of the active ingredients is 100%.

[0012] In the eutectoid gel as described above, the mass percentage of the deep eutectic solvent is 70%-80%, and the mass percentage of the lysozyme fiber is 20%-30%.

[0013] The second aspect of the present invention provides a method for preparing the eutectoid gel, comprising the following steps:

[0014] mixing choline chloride, gallic acid, and urea with the first aqueous solution, performing a first heating treatment, and stirring until a clear and transparent liquid is obtained to obtain a deep eutectic solvent;

[0015] The deep eutectic solvent and lysozyme fiber are mixed and then subjected to a second heating treatment, and stirred evenly to obtain the eutectic gel.

[0016] In the above-mentioned method for preparing the eutectoid gel, the mass of the first aqueous solution accounts for 8%-15% of the total mass of the choline chloride, the gallic acid and the urea.

[0017] The method for preparing the eutectoid gel as described above, wherein the temperature of the first heating treatment is 70-80°C;

[0018] The temperature of the second heating treatment is 60-80°C.

[0019] The preparation method of the eutectoid gel as described above, the preparation method of the lysozyme fiber comprises:

[0020] The lysozyme powder is dissolved in a second aqueous solution and dialyzed, and then lyophilized to obtain a purified protein;

[0021] Dissolving the purified protein in a third aqueous solution and adjusting the pH to 1.5-3 to obtain a protein solution;

[0022] The protein solution is transferred into a container and sealed, and then subjected to heating and stirring, quenching, and a second freeze-drying in sequence to obtain the lysozyme fiber.

[0023] The third aspect of the present invention provides a use of the eutectoid gel in preparing a drug for treating bacterial keratitis.

[0024] The fourth aspect of the present invention provides an antibacterial agent, which includes the eutectoid gel; the antibacterial agent can inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

[0025] The solution of the present invention has at least the following effects:

[0026] The eutectoid gel provided by the present invention comprises a deep eutectic solvent and lysozyme fibers. The eutectoid gel exhibits excellent anti-inflammatory and antibacterial properties, as well as corneal permeability. Studies have shown that the eutectoid gel has high DPPH and ABTS free radical scavenging abilities (anti-inflammatory properties), can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa (antibacterial properties), and has good corneal permeability. Therefore, it can be used to treat bacterial keratitis, providing a new theoretical basis and experimental foundation for the development of new drugs for the clinical treatment of bacterial keratitis, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0028] Figure 1 Schematic diagram of the preparation of the deep eutectic solvent and the eutectoid gel in Example 1 of the present invention;

[0029] Figure 2 The optical images of the deep eutectic solvent and the eutectoid gel in Examples 1-3 and Comparative Examples 1-2 of the present invention are shown in FIG. Figure 2 a is the optical image of the deep eutectic solvent (ChCl4:GA1) in Comparative Example 1, Figure 2b is the deep eutectic solvent (ChCl4:GA 0.75 :Urea 0.25 ), Figure 2 c is the deep eutectic solvent (ChCl4:GA 0.5 :Urea 0.5 ), Figure 2 d is the deep eutectic solvent (ChCl4:GA 0.25 :Urea 0.75 ), Figure 2 e is the optical image of the deep eutectic solvent (ChCl4:Urea1) in Comparative Example 2, Figure 2 f is the eutectoid gel (ChCl4:GA) in Example 1 0.75 :Urea 0.25 -Fly) optical image, where 1 is the eutectoid gel (ChCl4:GA) at the bottom of the sample bottle 0.75 :Urea 0.25 -Fly) optical images, Figure 2 g is the eutectoid gel (ChCl4:GA) in Example 2 0.5 :Urea 0.5 -Fly) optical image, where 2 is the eutectoid gel (ChCl4:GA) at the bottom of the sample bottle 0.5 :Urea 0.5 -Fly) optical images, Figure 2 h is the eutectoid gel (ChCl4:GA) in Example 3 0.25 :Urea 0.75 -Fly) optical image, where 3 is the eutectoid gel (ChCl4:GA) at the bottom of the sample bottle 0.25 :Urea 0.75 -Fly) optical images;

[0030] Figure 3 The NMR spectra of the deep eutectic solvents in Example 1, Examples 1-3, and Comparative Examples 1-2 of the present invention are shown;

[0031] Figure 4 IR spectra of lysozyme (Ly) and lysozyme fiber (Fly) in Example 1 of the present invention;

[0032] Figure 5 The scavenging rate of DPPH free radicals by the eutectoid gel in Examples 1-3 and Comparative Example 2 of the present invention;

[0033] Figure 6 The scavenging rate of ABTS free radicals by the eutectoid gel in Examples 1-3 and Comparative Example 2 of the present invention;

[0034] Figure 7The antibacterial performance test results of the coprecipitated gel in Examples 1-3 and Comparative Example 2 of the present invention are shown in FIG. Figure 7 a is a colony statistics diagram of Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) obtained by coprecipitation gel in Examples 1-3 and Comparative Example 2; Figure 7 b is the antibacterial rate of the coprecipitated gel in Examples 1-3 and Comparative Example 2 against Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli);

[0035] Figure 8 These are the permeability test results of the eutectoid gel (ChCl4:GA0.75:Urea0.25-Fly) in Example 1 of the present invention. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0037] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0038] As used herein, the term "treating" refers in some embodiments to ameliorating a disease or condition (i.e., slowing, arresting, or alleviating the development of a disease or condition or at least one of its clinical symptoms). In other embodiments, "treating" refers to alleviating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" refers to regulating a disease or condition physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a physical parameter), or both. In other embodiments, "treating" refers to preventing or delaying the onset, occurrence, or worsening of a disease or condition.

[0039] A first aspect of the present invention provides a eutectic gel comprising a deep eutectic solvent and lysozyme fibers;

[0040] The active ingredients of the deep eutectic solvent are composed of choline chloride, gallic acid and urea.

[0041] Specifically, the present invention first prepares a deep eutectic solvent using choline chloride, gallic acid, and urea as active ingredients. Then, using the deep eutectic solvent and lysozyme fiber as raw materials, a eutectic gel with excellent anti-inflammatory and antibacterial properties and corneal permeability is prepared. Research by the inventors has shown that the eutectic gel has high DPPH and ABTS free radical scavenging abilities, effectively inhibiting the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. It also has good corneal permeability, making it suitable for the treatment of bacterial keratitis.

[0042] In a specific embodiment, in the deep eutectic solvent, the mass percentage of choline chloride is 75%-85%, the mass percentage of gallic acid is 5%-15%, the mass percentage of urea is 5%-15%, and the sum of the active ingredients is 100%.

[0043] When the mass percentage of choline chloride, gallic acid, and urea in the deep eutectic solvent are each within the above ranges, the prepared eutectic gel has high DPPH free radical scavenging ability and ABTS free radical scavenging ability, and can inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0044] Furthermore, in the above-mentioned deep eutectic solvent, the mass percentage of choline chloride is 80%, the mass percentage of gallic acid is 10%-15%, the mass percentage of urea is 5%-10%, and the sum of the active ingredients is 100%. Preferably, the mass percentage of choline chloride is 80%, the mass percentage of gallic acid is 15%, the mass percentage of urea is 5%, and the sum of the active ingredients is 100%.

[0045] When the mass percentage of choline chloride, gallic acid, and urea in the deep eutectic solvent are each within the above ranges, the prepared eutectic gel has higher DPPH free radical scavenging ability and ABTS free radical scavenging ability, can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, and also has a good penetration effect on the cornea.

[0046] In a specific embodiment, in the eutectoid gel, the mass percentage of the deep eutectic solvent is 70%-80%, and the mass percentage of the lysozyme fiber is 20%-30%.

[0047] When the mass percentages of the deep eutectic solvent and the lysozyme fiber in the eutectic gel are respectively within the above ranges, the prepared eutectic gel has excellent anti-inflammatory properties, antibacterial properties and corneal permeability.

[0048] A second aspect of the present invention provides a method for preparing the above-mentioned eutectoid gel, comprising the following steps:

[0049] mixing choline chloride, gallic acid, and urea with the first aqueous solution, performing a first heating treatment, and stirring until a clear and transparent liquid is obtained to obtain a deep eutectic solvent;

[0050] The deep eutectic solvent and the lysozyme fiber are mixed and then subjected to a second heating treatment, and the eutectic gel is obtained after being stirred evenly.

[0051] The object prepared by the present invention is a eutectic gel. Specifically, choline chloride, gallic acid, and urea are mixed with a first aqueous solution to obtain a mixed solution, and then the mixed solution is subjected to a first heat treatment and stirred to a clear and transparent liquid to obtain a deep eutectic solvent; the deep eutectic solvent is mixed with lysozyme fiber to obtain a mixture, and the mixture is subjected to a second heat treatment and stirred to obtain a eutectic gel. The inventor's research shows that the eutectic gel prepared by this method has high DPPH free radical scavenging ability and ABTS free radical scavenging ability (anti-inflammatory performance), can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa (antibacterial performance), and it also has a good penetration effect on the cornea (corneal permeability). The method is simple in process, low in cost, and suitable for large-scale production.

[0052] The first aqueous solution may be any conventional water in the art. For example, the first aqueous solution may be deionized water.

[0053] The present invention does not particularly limit the specific equipment for performing the first and second heat treatments. For example, choline chloride, gallic acid, and urea can be mixed with the first aqueous solution and then placed in an oil bath for the first heat treatment.

[0054] In a specific embodiment, the mass of the first aqueous solution accounts for 8%-15% of the total mass of choline chloride, gallic acid and urea, for example, the mass of the first aqueous solution accounts for 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15% and the like of the total mass of choline chloride, gallic acid and urea.

[0055] When the mass of the first aqueous solution accounts for the total mass of choline chloride, gallic acid and urea within the above range, choline chloride, gallic acid and urea can be dissolved at a relatively low temperature (70-80° C.) to form a clear liquid.

[0056] In one embodiment, the temperature of the first heating treatment is 70-80°C.

[0057] When the temperature parameter of the first heat treatment is within the above range, the clear and transparent liquid can be dissolved to form a clear and transparent liquid without solvent precipitation.

[0058] For example, the temperature of the first heat treatment may be any one of 70° C., 72° C., 74° C., 76° C., 78° C., and 80° C., or any two of these.

[0059] In one embodiment, the temperature of the second heating treatment is 60-80°C.

[0060] When the temperature parameters of the second heating treatment are within the above range, the deep eutectic solvent can be better mixed with the lysozyme fiber, thereby obtaining a eutectic gel with a stable gel system (no solid precipitation phenomenon) and ensuring that the lysozyme fiber does not denature.

[0061] Illustratively, the temperature of the second heat treatment may be in the range of any one or any two of 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C, and 80°C.

[0062] In one embodiment, the preparation method of the lysozyme fiber comprises:

[0063] The lysozyme powder is dissolved in a second aqueous solution and then dialyzed, and a purified protein is obtained through a first freeze-drying process; the purified protein is dissolved in a third aqueous solution and the pH is adjusted to 1.5-3 to obtain a protein solution; the protein solution is transferred to a container and sealed, and then subjected to heating and stirring, quenching, and a second freeze-drying process in sequence to obtain lysozyme fibers.

[0064] The present invention adopts the above method to successfully prepare lysozyme fiber, and the lysozyme fiber can interact with the above deep eutectic solvent to prepare a eutectic gel with high DPPH free radical scavenging ability and ABTS free radical scavenging ability, which can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, and also has a good penetration effect on the cornea.

[0065] The second aqueous solution may be any conventional type in the art. For example, the second aqueous solution may be deionized water.

[0066] The third aqueous solution may be any conventional aqueous solution in the art. For example, the second aqueous solution may be pure water.

[0067] In a specific embodiment, the stirring temperature is 85-95° C., the rotation speed is 1000-3000 rpm, and the time is 4-10 h.

[0068] When the parameters of stirring temperature, rotation speed and time are each within the above ranges, lysozyme fibers can be obtained, which is beneficial for subsequently obtaining a eutectoid gel having a stable gel system by uniformly mixing the lysozyme fibers with a deep eutectic solvent.

[0069] Illustratively, the stirring temperature may be in the range of any one or any two of 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, and 95°C;

[0070] The rotation speed is any one of 1000 rpm, 1500 rpm, 2000 rpm, 2500 rpm, and 3000 rpm, or any two of them;

[0071] The time is any one of 4h, 5h, 6h, 7h, 8h, 9h, 10h, or a range consisting of any two of them.

[0072] A third aspect of the present invention provides the use of the aforementioned eutectoid gel in the preparation of a drug for treating bacterial keratitis. Research by the inventors has shown that the eutectoid gel provided by the present invention has excellent free radical scavenging ability, inhibiting the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. It also has good corneal penetration. Therefore, the eutectoid gel can be used to prepare a drug for treating bacterial keratitis.

[0073] A fourth aspect of the present invention provides an antibacterial agent comprising the aforementioned eutectoid gel; the antibacterial agent can inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. Research by the present inventors has shown that the eutectoid gel provided by the present invention can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, and therefore can be used to prepare an antibacterial agent.

[0074] Hereinafter, the solutions of the present invention will be described in detail through specific embodiments.

[0075] Example 1

[0076] Figure 1 Schematic diagram of the preparation of deep eutectic solvent (DES) and eutectoid gel in Example 1 of the present invention, as shown in FIG. Figure 1 As shown, the preparation method of the eutectoid gel of this embodiment includes the following steps:

[0077] Preparation of deep eutectic solvents

[0078] 1) Choline chloride (ChCl), gallic acid (GA), and urea (Urea) were added to deionized water in a mass ratio of 4:0.75:0.25 and mixed uniformly to obtain a mixed solution, wherein the mass of deionized water accounted for 10% of the total mass of ChCl, GA, and Urea;

[0079] 2) The mixed solution was placed in an oil bath at 70°C for heating and stirred until a clear and transparent liquid was obtained to obtain a deep eutectic solvent (ChCl4:GA 0.75 :Urea 0.25 ).

[0080] Preparation of lysozyme fiber

[0081] 1) Dissolve 10 g of lysozyme powder in 90 mL of deionized water to obtain a lysozyme solution;

[0082] 2) The lysozyme solution was transferred to a dialysis bag and dialyzed in a 4°C water bath for 3 days. The purified protein was obtained by lyophilization.

[0083] 3) Dissolve 223 mg of purified protein in 10.937 g of pure water to obtain a purified protein solution;

[0084] 4) adjusting the pH of the purified protein solution to 2 using hydrochloric acid to obtain a protein solution;

[0085] 5) Transfer the protein solution to a round-bottom flask, add a magnetic rod, seal the flask with sealing glue, place it in an oil bath, and heat and stir at 90°C and 2000 rpm for 8 h. After heating and stirring, quench it in an ice-water bath. Finally, transfer the quenched solution to a centrifuge tube and freeze-dry it to obtain lysozyme fibers.

[0086] Preparation of eutectoid gel

[0087] 1) mixing 1 g of deep eutectic solvent with 0.258 g of lysozyme fiber to obtain a mixture;

[0088] 2) The mixture was placed in a sample bottle, and then heated at 80°C and stirred to obtain a eutectic gel (ChCl4:GA 0.75 :Urea 0.25 -Fly).

[0089] Example 2

[0090] The preparation of the eutectoid gel provided in this embodiment is basically the same as that in Example 1, except that:

[0091] Preparation of deep eutectic solvents

[0092] 1) Choline chloride (ChCl), gallic acid (GA), and urea (Urea) were added to deionized water in a mass ratio of 4:0.5:0.5 and mixed evenly to obtain a mixed solution, wherein the mass of deionized water accounted for 10% of the total mass of ChCl, GA, and Urea;

[0093] 2) The mixed solution was placed in an oil bath at 70°C for heating and stirred until a clear and transparent liquid was obtained to obtain a deep eutectic solvent (ChCl4:GA 0.5 :Urea 0.5 ).

[0094] The preparation of the eutectoid gel in this embodiment is basically the same as that in embodiment 1, except that the deep eutectic solvent in this embodiment is used to prepare the eutectoid gel (ChCl4:GA 0.5 :Urea 0.5 -Fly).

[0095] Example 3

[0096] The preparation of the eutectoid gel provided in this embodiment is basically the same as that in Example 1, except that:

[0097] Preparation of deep eutectic solvents

[0098] 1) Choline chloride (ChCl), gallic acid (GA), and urea (Urea) were added to deionized water in a mass ratio of 4:0.25:0.75 and mixed evenly to obtain a mixed solution, wherein the mass of deionized water accounted for 10% of the total mass of ChCl, GA, and Urea;

[0099] 2) The mixed solution was placed in an oil bath at 70°C for heating and stirred until a clear and transparent liquid was obtained to obtain a deep eutectic solvent (ChCl4:GA 0.25 :Urea 0.75 ).

[0100] The preparation of the eutectoid gel in this embodiment is basically the same as that in embodiment 1, except that the deep eutectic solvent in this embodiment is used to prepare the eutectoid gel (ChCl4:GA 0.25 :Urea 0.75 -Fly).

[0101] Comparative Example 1

[0102] The preparation of the eutectoid gel provided in this comparative example is basically the same as that in Example 1, except that:

[0103] Preparation of deep eutectic solvents

[0104] 1) Choline chloride (ChCl) and gallic acid (GA) were added to deionized water in a mass ratio of 4:1 and mixed evenly to obtain a mixed solution, wherein the mass of deionized water accounted for 10% of the total mass of ChCl and GA;

[0105] 2) The mixed solution was placed in an oil bath at 70°C for heating and stirred until a clear liquid was obtained to obtain a deep eutectic solvent (ChCl4:GA1).

[0106] The preparation of the eutectoid gel in this comparative example is basically the same as that in Example 1, except that the deep eutectic solvent in this example is used to prepare the eutectoid gel (ChCl4:GA1-Fly).

[0107] Figure 2a in the figure is an optical image of the deep eutectic solvent (ChCl4:GA1) in Comparative Example 1.

[0108] Depend on Figure 2 It can be seen from a that a large amount of solid precipitated from the deep eutectic solvent (ChCl4:GA1) in Comparative Example 1, which was turbid, indicating that the performance of the deep eutectic solvent (ChCl4:GA1) itself was unstable. The eutectic gel (ChCl4:GA1-Fly) prepared using it also had a large amount of solid precipitated. Therefore, no subsequent performance test was performed on the eutectic gel (ChCl4:GA1-Fly).

[0109] Comparative Example 2

[0110] The preparation of the eutectoid gel provided in this comparative example is basically the same as that in Example 1, except that:

[0111] Preparation of deep eutectic solvents

[0112] 1) Choline chloride (ChCl) and urea (Urea) were added to deionized water in a mass ratio of 4:1 and mixed evenly to obtain a mixed solution, wherein the mass of deionized water accounted for 10% of the total mass of ChCl and GA;

[0113] 2) The mixed solution was placed in an oil bath at 70°C for heating and stirred until a clear and transparent liquid was obtained to obtain a deep eutectic solvent (ChCl4:Urea1).

[0114] The preparation of the eutectoid gel in this comparative example is basically the same as that in Example 1, except that the deep eutectic solvent in this example is used to prepare the eutectoid gel (ChCl4:Urea1-Fly).

[0115] Performance Testing

[0116] 1. At room temperature, the deep eutectic solvents and eutectoid gels in Examples 1-3 of the present invention and the deep eutectic solvents in Comparative Examples 1-2 of the present invention were photographed respectively. The results are as follows: Figure 2 shown.

[0117] Depend on Figure 2 It can be seen from a that a large amount of solid precipitated in the deep eutectic solvent (ChCl4:GA1) in Comparative Example 1, which was turbid.

[0118] Depend on Figure 2 It can be seen from bd that the deep eutectic solvents in Examples 1-3 of the present invention are clear and transparent liquids, which proves that the deep eutectic solvents are successfully prepared.

[0119] Depend on Figure 2 It can be seen from the figure that a small amount of solid precipitated from the deep eutectic solvent (ChCl4:Urea1) in Comparative Example 2, which was turbid.

[0120] Depend on Figure 2 It can be seen from fh that the eutectoid gels in Examples 1-3 of the present invention are non-flowing gels and no solid is precipitated, which indicates that the eutectoid gels in Examples 1-3 of the present invention have stable gel systems.

[0121] 2. The molecular structures and intermolecular interactions of the deep eutectic solvents in Examples 1-3 and Comparative Examples 1-2 were measured using a nuclear magnetic resonance spectrometer (Bruker AVIII HD 400 MHz). Figure 3 shown.

[0122] Depend on Figure 3 It can be seen that the methyl peak of choline chloride (ChCl) appears at δ3.1ppm, the methylene peak of choline chloride at δ3.8ppm, the hydroxyl peak of choline chloride superimposed with the amino peak of urea (Urea) at δ5.5ppm, and the proton on the aromatic ring of gallic acid (GA) at δ6.9ppm, indicating the successful synthesis of the deep eutectic solvents in Examples 1-3 of the present invention and Comparative Examples 1-2. In addition, the hydroxyl (-OH) protons of ChCl and the amino (-NH2) protons of Urea show changes in chemical shifts, indicating the presence of hydrogen bonding interactions between molecules within the deep eutectic solvents in Examples 1-3 of the present invention and Comparative Example 2.

[0123] 3. Infrared spectroscopy tests were performed on the lysozyme (Ly) and lysozyme fiber (Fly) in Example 1 of the present invention. Figure 4 shown.

[0124] Depend on Figure 4 It can be seen that lysozyme (Ly) is fibrillated, and the present invention successfully prepares lysozyme fiber (Fly).

[0125] 4. The free radical scavenging ability of the eutectoid gels in Examples 1-3 of the present invention and Comparative Example 2 was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) reagent and 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) reagent, respectively, to illustrate the anti-inflammatory properties of the eutectoid gels.

[0126] (1) DPPH free radical scavenging experiment, the method is as follows:

[0127] 1 mg of DPPH reagent was mixed with 25 mL of methanol and stirred at 37 °C in the dark for 12 h to obtain a DPPH stock solution;

[0128] Take 5 mL of DPPH stock solution and mix it with 5 mL of deionized water to obtain DPPH dilution solution;

[0129] The eutectoid gel sample was mixed with deionized water to prepare a eutectoid gel sample with a concentration of 20 μg / mL;

[0130] 100 μL of DPPH dilution was mixed with 100 μL of 20 μg / mL coprecipitate gel sample, and then cultured in a constant temperature incubator at 37°C. The absorbance at 734 nm was measured at 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min, and 120 min, and the DPPH free radical scavenging rate was calculated.

[0131] The eutectoid gels in Examples 1-3 of the present invention and Comparative Example 2 were used as eutectoid gel samples, and DPPH free radical scavenging experiments were carried out according to the above method. The results are as follows: Figure 5 As shown; Figure 5 It is the scavenging rate of DPPH free radicals by the eutectoid gel in Examples 1-3 of the present invention and Comparative Example 2.

[0132] Depend on Figure 5 It can be seen that the DPPH radical scavenging rates of the eutectoid gels in Examples 1-3 of the present invention are all higher than that of the eutectoid gel in Comparative Example 2.

[0133] (2) ABTS free radical scavenging experiment, the method is as follows:

[0134] Mix 54.04 mg of ABTS reagent, 9.93 mg of potassium persulfate, and 15 mL of water and stir at 37°C in the dark for 12 hours to obtain an ABTS stock solution;

[0135] Take 5 mL of ABTS stock solution and mix it with 5 mL of deionized water to obtain ABTS dilution solution;

[0136] The eutectoid gel sample was mixed with deionized water to prepare a eutectoid gel sample with a concentration of 20 μg / mL;

[0137] Take 100 μL of ABTS dilution solution and mix it with 100 μL of 20 μg / mL coprecipitate gel sample, then incubate it in a constant temperature incubator at 37°C. The absorbance at 734 nm was measured at 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 60 min, and 120 min, and the ABTS free radical scavenging rate was calculated.

[0138] The eutectoid gels in Examples 1-3 of the present invention and Comparative Example 2 were used as eutectoid gel samples, and ABTS free radical scavenging experiments were carried out according to the above method. The results are as follows: Figure 6 As shown; Figure 6 The scavenging rates of the eutectoid gels for ABTS free radicals in Examples 1-3 and Comparative Example 2 of the present invention are shown.

[0139] Depend on Figure 6It can be seen that the ABTS radical scavenging rates of the eutectoid gels in Examples 1-3 of the present invention are all higher than that of the eutectoid gel in Comparative Example 2.

[0140] The above results show that the eutectoid gel provided by the embodiment of the present invention has high DPPH free radical scavenging ability and ABTS free radical scavenging ability, which illustrates that the eutectoid gel provided by the embodiment of the present invention has excellent anti-inflammatory properties.

[0141] 5. The antibacterial performance of the eutectoid gels in Examples 1-3 of the present invention and Comparative Example 2 was tested respectively. The test method is as follows:

[0142] (1) Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Escherichia coli (E. coli) stored in a -80°C ultra-low temperature refrigerator were taken out and placed on a clean bench. After each strain was thawed, the thawed strains were taken out using a sterilized inoculation ring and placed into 20 mL of Luria-Bertani (LB) medium. The culture was carried out at 37°C and 150 rpm overnight to obtain P. aeruginosa, S. aureus, and E. coli bacterial solutions, respectively. The OD values of the P. aeruginosa, S. aureus, and E. coli bacterial solutions were measured using a microplate reader. 600 value, and dilute them to obtain a concentration of 10 6 CFU / mL of P. aeruginosa bacterial solution, concentration of 10 6 CFU / mL of S. aureus bacterial solution and a concentration of 10 6 CFU / mL of E. coli liquid.

[0143] (2) Add 0.05 g of the eutectoid gel to be tested into a 24-well plate, and then add 1.95 mL of a 10% 6 CFU / mL of P. aeruginosa was used as the experimental group, and the control group was added with 0.05mL PBS and 1.95mL of 10 6 CFU / mL of P. aeruginosa bacterial solution was placed in a 37°C incubator for 12 hours to obtain the incubated P. aeruginosa bacterial solution. The OD values of the P. aeruginosa bacterial solution before and after incubation in the experimental group and the control group were measured using a microplate reader. 600 After the measurement, the incubated P. aeruginosa bacterial solution was diluted to obtain a concentration of 10 3 CFU / mL of incubated P. aeruginosa bacterial solution, and then 100 μL of 10 CFU / mL ...3 After incubation with a concentration of 100 CFU / mL of P. aeruginosa bacterial solution, evenly spread it on the agar plate with a disposable coating stick, and incubate it in a 37°C incubator for 18 hours. Finally, place the agar plate under a colony counter and take pictures to observe the colony distribution of P. aeruginosa; according to the OD value of the P. aeruginosa bacterial solution before incubation, 600 The OD of the P. aeruginosa bacterial solution after incubation 600 The inhibition rate of the tested eutectoid gel on P. aeruginosa was calculated using the following formula:

[0144]

[0145] Among them, OD 0样品 represents the OD of P. aeruginosa bacterial solution before incubation in the experimental group 600 Value, OD 1样品 Indicates the OD of the P. aeruginosa bacterial solution after incubation in the experimental group 600 Value, OD 0空白 represents the OD of the P. aeruginosa bacterial solution before incubation in the control group 600 Value, OD 1空白 Indicates the OD of the P. aeruginosa bacterial solution after incubation in the control group 600 value.

[0146] (3) Add 0.05 g of the eutectoid gel to be tested into a 24-well plate, and then add 1.95 mL of a 10% 6 CFU / mL of S. aureus was used as the experimental group, and the control group (control) was added with 0.05mL PBS and 1.95mL of 10 6 CFU / mL of S. aureus bacterial solution was placed in a 37°C incubator for 12 hours to obtain the incubated S. aureus bacterial solution. The OD of the S. aureus bacterial solution before and after incubation in the experimental group and the control group were measured using a microplate reader. 600 After the measurement, the incubated S. aureus bacterial solution was diluted to obtain a concentration of 10 3 CFU / mL of incubated S. aureus bacteria solution, and then 100 μL of 10 CFU / mL ... 3 After incubation, the S. aureus bacterial solution with a CFU / mL was evenly spread on the agar plate with a disposable coating stick, and then placed in a 37°C incubator for 18 hours. Finally, the agar plate after incubation was placed under a colony counter and photographed to observe the colony distribution of S. aureus; according to the OD of the S. aureus bacterial solution before incubation,600 The OD of the S. aureus bacterial solution after incubation 600 The inhibition rate of the co-precipitated gel to S. aureus was calculated using the following formula:

[0147]

[0148] Among them, OD 0样品 Indicates the OD of the S. aureus bacterial solution before incubation in the experimental group 600 Value, OD 1样品 Indicates the OD of the S. aureus bacterial solution after incubation in the experimental group 600 Value, OD 0空白 Indicates the OD of the S. aureus bacterial solution before incubation in the control group 600 Value, OD 1空白 Indicates the OD of the S. aureus bacterial solution after incubation in the control group 600 value.

[0149] (4) Add 0.05 g of the eutectoid gel to be tested into a 24-well plate, and then add 1.95 mL of a 10% 6 CFU / mL of E. coli liquid was used as the experimental group, and the control group (control) was added with 0.05mL PBS and 1.95mL of 10 6 The E. coli bacterial solution with a CFU / mL was placed in a 37°C incubator for 12 hours to obtain the incubated E. coli bacterial solution. The OD of the E. coli bacterial solution before and after incubation in the experimental group and the control group were measured using a microplate reader. 600 After the measurement, the incubated E. coli solution was diluted to obtain a concentration of 10 3 CFU / mL of incubated E. coli solution, and then 100 μL of 10 CFU / mL of 10 3 After incubation, the E. coli liquid was evenly spread on the agar plate with a disposable coating stick and incubated in a 37°C incubator for 16 hours. Finally, the agar plate was placed under a colony counter and photographed to observe the distribution of E. coli colonies. According to the OD value of the E. coli liquid before incubation, the plate was placed under a colony counter to observe the distribution of E. coli colonies. 600 The OD value of the E. coli culture after incubation 600 The inhibition rate of the co-precipitated gel to E. coli was calculated using the following formula:

[0150]

[0151] Among them, OD 0样品 Indicates the OD of E.coli culture before incubation in the experimental group 600Value, OD 1样品 Indicates the OD of the E.coli culture solution after incubation in the experimental group 600 Value, OD 0空白 Indicates the OD of the E. coli culture solution before incubation in the control group 600 Value, OD 1空白 Indicates the OD of the E.coli culture solution after incubation in the control group 600 value.

[0152] The eutectoid gels in Examples 1-3 of the present invention and Comparative Example 2 were used as the eutectoid gels to be tested, and the antibacterial performance tests were performed according to the above test method. The results are as follows: Figure 7 As shown; Figure 7 The antibacterial performance test results of the coprecipitated gel in Examples 1-3 and Comparative Example 2 of the present invention are shown in FIG. Figure 7 a is a colony statistics diagram of Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) obtained by coprecipitation gel in Examples 1-3 and Comparative Example 2; Figure 7 b is the antibacterial rate of the coprecipitated gel in Examples 1-3 and Comparative Example 2 against Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli).

[0153] Depend on Figure 7 It can be seen from a that compared with the control group, the co-clustered gel in Examples 1-2 can effectively inhibit the growth of Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S.aureus) and Escherichia coli (E.coli), the co-clustered gel in Example 3 of the present invention can effectively inhibit the growth of Pseudomonas aeruginosa (P.aeruginosa) and Staphylococcus aureus (S.aureus), while the co-clustered gel in Comparative Example 2 can only effectively inhibit the growth of Staphylococcus aureus (S.aureus), and from Figure 7 As can be seen from a, in Example 1, the eutectoid gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) has better antibacterial performance than the coprecipitated gel in Examples 2-3 and Comparative Example 2.

[0154] from Figure 7 The a can be obtained, the eutectoid gel (ChCl4:GA 0.75 :Urea 0.25-Fly) has excellent antibacterial effect on Pseudomonas aeruginosa (P.aeruginosa) and Staphylococcus aureus (S.aureus), the main pathogens of bacterial keratitis; the broad-spectrum antibacterial effect of the co-cluster gel was tested with Escherichia coli (E.coli), and it was found that the co-cluster gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) also has excellent antibacterial effect on Escherichia coli (E. coli).

[0155] Depend on Figure 7 As can be seen from b, the eutectoid gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) can achieve an inhibition rate of 96% against Pseudomonas aeruginosa (P.aeruginosa) and 94% against Staphylococcus aureus (S.aureus). It can also effectively inhibit the growth of Escherichia coli (E.coli) with an inhibition rate of up to 100%.

[0156] The above results show that the co-clustered gels in Examples 1-3 of the present invention can effectively inhibit the growth of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus), the main pathogens of bacterial keratitis, and have excellent antibacterial properties, and the co-clustered gel in Example 1 has the best antibacterial property.

[0157] 6. Based on Examples 2-3 and Comparative Example 2, the anti-inflammatory and antibacterial properties of the coprecipitated gel (ChCl4:GA) in Example 1 were both weaker than those of the coprecipitated gel (ChCl4:GA) in Example 1. 0.75 :Urea 0.25 -Fly), so the coprecipitation gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) was used as the experimental group for penetration performance test, and the test method was as follows:

[0158] 4.79 mg of Rhodamine B was mixed with 1 g of deep eutectic solvent (ChCl4:GA 0.75 :Urea 0.25 ) to ensure that Rhodamine B is evenly distributed to obtain a deep eutectic solvent containing Rhodamine B, and then the eutectic gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) was immersed in a deep eutectic solvent containing rhodamine B for 30 min to perform solvent replacement to obtain a eutectic gel after immersion;

[0159] The cornea was isolated from the bovine eye, washed in PBS, and then fixed on a circular mold with the epithelium facing up. The soaked eutectoid gel was then placed on the corneal surface as the experimental group (ChCl4:GA 0.75 :Urea 0.25 -Fly), and the control group (PBS) was cleaned with PBS only. The corneas placed with the soaked eutectoid gel or the cleaned corneas were then incubated at 37°C for 5 min, 10 min, 15 min, 30 min, and 60 min, respectively. After the incubation, the corneal surface was gently rinsed with PBS, and the excess liquid was absorbed with filter paper. The cornea and the mold were placed on the stage of a confocal laser microscope to ensure that the corneal surface was smooth and the epithelium was facing upward. 540 nm was selected as the excitation wavelength and the emission filter was 570 nm. Starting from the corneal surface, tomography was performed along the Z-axis direction to obtain fluorescence images of different penetration depths (such as Figure 8 The deeper the penetration depth, the better the penetration effect.

[0160] Depend on Figure 8 It can be seen that as time goes by, the eutectoid gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) has a better penetration effect on the cornea, and the penetration depth is much higher than that of the control group (PBS), which shows that the eutectoid gel (ChCl4:GA 0.75 :Urea 0.25 -Fly) has a good penetration effect on the cornea.

[0161] In summary, the eutectoid gel provided in Example 1 of the present invention has high DPPH free radical scavenging ability and ABTS free radical scavenging ability (anti-inflammatory property), can effectively inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa (antibacterial property), and it also has a good penetration effect on the cornea (corneal permeability), and can be used to treat bacterial keratitis, providing a new theoretical basis and experimental basis for the development of new drugs for the clinical treatment of bacterial keratitis, and has broad application prospects.

[0162] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A eutectoid gel, characterized in that It includes deep eutectic solvent and lysozyme fibers; The effective components of the deep eutectic solvent consist of choline chloride, gallic acid and urea.

2. The eutectoid gel according to claim 1, characterized in that In the deep eutectic solvent, the mass percentage of the choline chloride is 75%-85%, the mass percentage of the gallic acid is 5%-15%, the mass percentage of the urea is 5%-15%, and the sum of the active ingredients is 100%.

3. The eutectoid gel according to claim 2, characterized in that In the deep eutectic solvent, the mass percentage of the choline chloride is 80%, the mass percentage of the gallic acid is 10%-15%, the mass percentage of the urea is 5%-10%, and the sum of the effective ingredients is 100%.

4. The eutectoid gel according to claim 1, characterized in that In the eutectoid gel, the mass percentage of the deep eutectic solvent is 70%-80%, and the mass percentage of the lysozyme fiber is 20%-30%.

5. A method for preparing the eutectoid gel according to any one of claims 1 to 4, characterized in that: The following steps are involved: mixing choline chloride, gallic acid, and urea with the first aqueous solution, performing a first heating treatment, and stirring until a clear and transparent liquid is obtained to obtain a deep eutectic solvent; The deep eutectic solvent and lysozyme fiber are mixed and then subjected to a second heating treatment, and stirred evenly to obtain the eutectic gel.

6. The method for preparing the eutectoid gel according to claim 5, characterized in that: The mass of the first aqueous solution accounts for 8%-15% of the total mass of the choline chloride, the gallic acid and the urea.

7. The method for preparing the eutectoid gel according to claim 5, wherein: The temperature of the first heating treatment is 70-80°C; The temperature of the second heating treatment is 60-80°C.

8. The method for preparing the eutectoid gel according to claim 5, wherein: The preparation method of the lysozyme fiber comprises: The lysozyme powder is dissolved in a second aqueous solution and dialyzed, and then lyophilized to obtain a purified protein; Dissolving the purified protein in a third aqueous solution and adjusting the pH to 1.5-3 to obtain a protein solution; The protein solution is transferred into a container and sealed, and then subjected to heating and stirring, quenching, and a second freeze-drying in sequence to obtain the lysozyme fiber.

9. Use of the eutectoid gel according to any one of claims 1 to 4 in preparing a drug for treating bacterial keratitis.

10. An antibacterial agent, characterized in that The invention comprises the eutectoid gel according to any one of claims 1 to 4; the antibacterial agent can inhibit the growth of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa.

Images