Self-assembly eutectoid gel with cell permeability and multiple anti-inflammatory effects as well as preparation method and application of self-assembly eutectoid gel

Abstract

The invention belongs to the technical field of biomedical materials, and discloses a self-assembly eutectoid gel with cell permeability and multiple anti-inflammatory effects and a preparation method of the self-assembly eutectoid gel. The preparation method comprises the following steps: mixing and heating choline chloride and a hydroxyl type hydrogen bond donor to obtain viscous liquid; dissolving the viscous liquid in water to prepare a DES solution; adding antibacterial active ingredients and lysozyme fibers into the DES solution, heating, carrying out ultrasonic treatment, and standing to room temperature, so as to obtain the self-assembled eutectoid gel. According to the application, the deep eutectic solvent with high biocompatibility, solubility promotion and cell permeability promotion is used as a solvent of the eutectoid gel to prepare the self-assembled eutectoid gel, so that the drug utilization efficiency of small molecule drugs in the eutectoid gel is enhanced, intracellular active oxygen can be effectively removed, and macrophages of M1 pro-inflammatory phenotype are polarized into M2 anti-inflammatory phenotype. The preparation method meets the preparation standard of green chemistry, and is safe and environment-friendly.

Description

Technical Field

[0001] The present application belongs to the technical field of biomedical materials, and specifically relates to a self-assembled co-evolution gel with cell permeability and multiple anti-inflammatory effects and a preparation method thereof. Background Art

[0002] Deep eutectic solvents (DES) consist of at least one hydrogen bond acceptor (HBA) and one hydrogen bond donor (HBD), which exist in the eutectic mixture primarily through hydrogen bonds when these compounds are mixed in a specific molar ratio. DES have attracted extensive attention in many industries such as material synthesis, catalytic reactions, electrochemistry, biomaterial separation and extraction due to their unique properties, such as excellent thermal stability, adjustable structural characteristics, high solubility for a variety of solutes, and facile preparation methods, economies of scale, and green sustainable development. In addition, DES also has strong biodegradability and biocompatibility, as well as an excellent ability to accelerate drug solubility and permeability, thereby improving the bioavailability of many drugs. The inherent properties of DES have the potential to address the limitations of traditional hydrogels in terms of diffusivity and permeability, especially for molecules with low water solubility.

[0003] At present, eutectoid gels with DESs as the effective liquid active ingredient are attractive choices in various industrial fields due to their remarkable stability, cost-effectiveness and environmental friendliness, and have been widely used in sensing, conductivity and interface lubrication. However, the application potential of eutectoid gels containing DES in inflammatory diseases such as chronic periodontitis has not been fully explored.

[0004] Based on this, there is an urgent need to develop a self-assembled co-evolution gel with cell permeability and multiple anti-inflammatory effects, broaden the application range of the co-evolution gel containing DES, and apply it to the treatment of inflammatory diseases such as chronic periodontitis. Summary of the invention

[0005] The present application provides a self-assembled co-evolution gel with cell permeability and multiple anti-inflammatory effects and a preparation method thereof, aiming to develop a co-evolution gel containing DES that can be used for inflammatory diseases.

[0006] In order to achieve the above objectives, the present application adopts the following technical solutions.

[0007] In a first aspect of the present application, a method for preparing a self-assembled co-clustered gel having cell permeability and multiple anti-inflammatory effects is provided, comprising:

[0008] S1, mixing choline chloride and a hydroxyl type hydrogen bond donor and heating them to obtain a viscous liquid; dissolving the viscous liquid in water to prepare a DES solution;

[0009] S2, adding antibacterial active ingredients and lysozyme fibers to the DES solution, heating and ultrasonic treatment, and standing to room temperature to obtain a self-assembled eutectoid gel.

[0010] Preferably, the hydroxyl type hydrogen bond donor is mannose or xylitol.

[0011] Preferably, the antibacterial active ingredient is gallic acid or epigallocatechin gallate.

[0012] Preferably, the molar ratio of the choline chloride to the hydroxyl type hydrogen bond donor is (4:1) to (1:4).

[0013] Preferably, the concentration of the DES solution is 5 to 90 wt %.

[0014] Preferably, the amount of the antibacterial active ingredient added to the DES solution is 10-50 mg / mL.

[0015] Preferably, the amount of the lysozyme fiber added to the DES solution is 10-50 mg / mL.

[0016] Preferably, the heating temperature in S1 is 50-90°C; and in S2, the heating temperature is 30-50°C.

[0017] The second aspect of the present application provides a self-assembled co-evolution gel having cell permeability and multiple anti-inflammatory effects prepared by the above preparation method.

[0018] The third aspect of the present application provides the use of the self-assembled co-clustered gel in drugs for treating inflammatory diseases.

[0019] Compared with the prior art, the beneficial effects of this application are:

[0020] The present application prepares a self-assembled eutectoid gel by using a deep eutectic solvent with high biocompatibility, solubility-promoting and cell-permeability-promoting properties as the eutectoid gel solvent, thereby enhancing the drug utilization efficiency of small molecule drugs in the eutectoid gel, being able to effectively remove intracellular reactive oxygen species, and polarize macrophages with an M1 pro-inflammatory phenotype to an M2 anti-inflammatory phenotype. The eutectoid gel is applied to drugs for inflammatory diseases, and has a good therapeutic effect on inflammatory diseases.

[0021] The preparation method of the present application complies with the preparation standards of green chemistry and is safe and environmentally friendly. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 Self-assembled eutectoid gel DES 10 -FLy 30 -GA 30 Physical picture;

[0024] Figure 2 The rheological curves of the self-assembled eutectoid gel prepared in Example and Comparative Example 1;

[0025] Figure 3 Self-assembled eutectoid gel DES 10 -FLy 30 -GA 30 Colony statistics of Escherichia coli and Staphylococcus aureus;

[0026] Figure 4 Self-assembled eutectoid gel DES 10 -FLy 30 -GA 30 And the macrophage flow cytometry graphs of the coprecipitation gels prepared in Comparative Examples 1-3. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0028] In the following description of this embodiment, the terms "include", "comprising", "having" and "containing" are all open terms, meaning including but not limited to.

[0029] In the following description of this embodiment, the term "and / or" is used to describe the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, B exists alone, and A and B exist at the same time. A and B can be singular or plural. The character " / " generally indicates that the associated objects before and after are in an "or" relationship.

[0030] In the following description of this embodiment, the term "at least one" refers to one or more, and "plurality" refers to two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b or c", or "at least one of a, b and c", can all represent: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0031] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0032] Those skilled in the art should understand that in the following description of the embodiments of the present application, the order of serial numbers does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0033] Those skilled in the art will appreciate that the numerical ranges in the embodiments of the present application are to be construed as specifically disclosing each intermediate value between the upper and lower limits of the scope. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded in the scope.

[0034] Unless otherwise specified, the technical / scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this application belongs. Although this application only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of this application. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0035] In a first aspect, the present application provides a method for preparing a self-assembled co-clustered gel having cell permeability and multiple anti-inflammatory effects, comprising:

[0036] S1, mixing choline chloride and a hydroxyl type hydrogen bond donor and heating them to obtain a viscous liquid; dissolving the viscous liquid in water to prepare a DES solution;

[0037] In the present application, the hydroxyl type hydrogen bond donor is mannose or xylitol; preferably, the molar ratio of the choline chloride to the hydroxyl type hydrogen bond donor is (4:1) to (1:4).

[0038] The present invention mixes choline chloride and hydroxyl hydrogen bond donor and heats to 50-90° C. to obtain a viscous liquid. In the DES solution prepared in the present invention, the sum of the mass concentrations of choline chloride and hydroxyl hydrogen bond donor in the DES solution is 5-90 wt %.

[0039] S2, adding antibacterial active ingredients and lysozyme fibers to the DES solution, heating and ultrasonic treatment, and standing to room temperature to obtain a self-assembled eutectoid gel.

[0040] In the present application, the antibacterial active ingredient is gallic acid or epigallocatechin gallate. The DES solution to which the antibacterial active ingredient and lysozyme fiber are added is heated to 30-50°C and allowed to stand at room temperature to obtain a self-assembled eutectoid gel. Preferably, the amount of the antibacterial active ingredient added to the DES solution is 10-50 mg / mL; the amount of the lysozyme fiber added to the DES solution is 10-50 mg / mL.

[0041] The preparation method of the present application complies with the preparation standards of green chemistry and is safe and environmentally friendly. The present application prepares a self-assembled eutectoid gel by using a deep eutectic solvent with high biocompatibility, solubility-promoting properties and cell permeability-promoting properties as an eutectoid gel solvent, thereby enhancing the drug utilization efficiency of small molecule drugs in the eutectoid gel.

[0042] In the second aspect, the present application provides a self-assembled co-evolution gel prepared by the above-mentioned preparation method. The self-assembled co-evolution gel has cell permeability and multiple anti-inflammatory effects, can effectively remove intracellular reactive oxygen species, and polarize macrophages with M1 pro-inflammatory phenotype to M2 anti-inflammatory phenotype, and is suitable for inflammatory diseases. The self-assembled co-evolution gel with cell permeability and multiple anti-inflammatory effects prepared in the present application can be used to prepare drugs for the treatment of inflammatory diseases, such as drugs for periodontitis.

[0043] The present application is further described below by way of examples.

[0044] Example 1

[0045] This embodiment provides a method for preparing a self-assembled eutectoid gel, comprising:

[0046] S1, add 2.187g choline chloride and 2.813g mannose to a 50mL centrifuge tube; add a magnet to the centrifuge tube, place the centrifuge tube in an 80°C oil bath, and stir until a clear, transparent, viscous liquid is formed. Add 45mL deionized water to the liquid to obtain a DES solution with a concentration of 10wt%.

[0047] S2, 30 mg / mL lysozyme fibrils and 30 mg / mL gallic acid were dissolved in 1 mL of 10 wt% DES solution, and ultrasonic treatment was performed at 37°C. After standing for 10 minutes, a self-assembled eutectoid gel was obtained, which was recorded as DES 10 -FLy 30 -GA 30 .

[0048] Example 2

[0049] This embodiment provides a method for preparing a self-assembled eutectoid gel, comprising:

[0050] S1, add 2.187g choline chloride and 2.813g mannose to a 50mL centrifuge tube; add a magnet to the centrifuge tube, place the centrifuge tube in an 80°C oil bath, and stir until a clear, transparent, viscous liquid is formed. Add 45mL deionized water to the liquid to obtain a DES solution with a concentration of 10wt%.

[0051] S2, 20 mg / mL lysozyme fibrils and 40 mg / mL gallic acid were dissolved in 1 mL of 10 wt% DES solution, and ultrasonic treatment was performed at 37°C. After standing for 10 minutes, a self-assembled eutectoid gel was obtained, which was recorded as DES 10 -FLy 20 -GA 40 .

[0052] Example 3

[0053] This embodiment provides a method for preparing a self-assembled eutectoid gel, comprising:

[0054] S1, add 2.187g choline chloride and 2.813g mannose to a 50mL centrifuge tube; add a magnet to the centrifuge tube, place the centrifuge tube in an 80°C oil bath, and stir until a clear, transparent, viscous liquid is formed. Add 45mL deionized water to the liquid to obtain a DES solution with a concentration of 10wt%.

[0055] S2, 40 mg / mL lysozyme fibrils and 20 mg / mL gallic acid were dissolved in 1 mL of 10 wt% DES solution, and ultrasonic treatment was performed at 37°C. After standing for 10 minutes, a self-assembled eutectoid gel was obtained, which was recorded as DES 10 -FLy 40 -GA 20 .

[0056] Comparative Example 1

[0057] S1, add 2.187g choline chloride and 2.813g mannose to a 50mL centrifuge tube; add a magnet to the centrifuge tube, place the centrifuge tube in an 80°C oil bath, and stir until a clear, transparent, viscous liquid is formed. Add 45mL deionized water to the liquid to obtain a DES solution with a concentration of 10wt%.

[0058] S2, 30 mg / mL lysozyme fibrils were dissolved in 1 mL of 10 wt% DES solution, and ultrasonic treatment was performed at 37°C. After standing for 10 minutes, a co-precipitate gel was obtained, which was recorded as DES 10 -FLy 30 .

[0059] Comparative Example 2

[0060] S1, add 2.187g choline chloride and 2.813g mannose to a 50mL centrifuge tube; add a magnet to the centrifuge tube, place the centrifuge tube in an 80°C oil bath, and stir until a clear, transparent, viscous liquid is formed. Add 45mL deionized water to the liquid to obtain a DES solution with a concentration of 10wt%.

[0061] S2, 30 mg / mL gallic acid was dissolved in 1 mL of 10 wt% DES solution, and ultrasonic treatment was performed at 37°C. After standing for 10 minutes, a co-precipitate gel was obtained, which was recorded as DES 10 -GA 30 .

[0062] Comparative Example 3

[0063] 30 mg / mL lysozyme fibrils and 30 mg / mL gallic acid were dissolved in 1 mL deionized water, ultrasonically treated at 37 °C, and allowed to stand for 10 minutes to obtain a solution, which was recorded as FLy 30 -GA 30 .

[0064] Self-assembled eutectoid gel DES prepared in Example 1 10 -FLy 30 -GA 30 , the actual picture is as follows Figure 1 As shown. Figure 1 It can be seen that after the sample bottle was inverted, the gel did not flow, proving that the self-assembled eutectoid gel was successfully prepared.

[0065] The self-assembled eutectoid gel DES prepared in Examples 1-3 10 -FLy 30 -GA 30 、DES 10 -FLy 20 -GA 40 and DES 10 -FLy40 -GA 20 , and the eutectoid gel DES prepared in Comparative Example 1 10 -FLy 30 The rheological properties were tested by using a rheometer with an oscillating mode to perform a shear rheological test on the eutectic gel, with a shear strain of 1% and an angular frequency of ω in the range of 0.1-100 rad s -1 The changes are logarithmic.

[0066] Test results such as Figure 2 As shown. Figure 2 It can be seen that when the angular velocity is less than 100 rad s -1 , the storage modulus is greater than the loss modulus, and the higher the Fly content, the larger the storage modulus; this indicates that Fly provides support for the gel network, and DES can stabilize the fibrotic conformation of Fly and stabilize the gel network.

[0067] The self-assembled eutectoid gel DES prepared in Example 1 10 -FLy 30 -GA 30 The antibacterial performance test of Staphylococcus aureus and Escherichia coli was carried out as follows:

[0068] Take out Staphylococcus aureus and Escherichia coli stored in -80℃ ultra-low temperature refrigerator and put them into the clean bench. After the strains are thawed, use a sterilized inoculation ring to take the frozen bacteria block in the test tube and put it into 20mL Luria-Bertani (LB) medium. Incubate at 37℃ and 150rpm overnight to obtain Staphylococcus aureus and Escherichia coli liquids. Use a microplate reader to measure the OD of Staphylococcus aureus and Escherichia coli liquids. 630 , and dilute the Staphylococcus aureus and Escherichia coli cultures to a concentration of 10 6 CFU / mL is reserved.

[0069] The self-assembled eutectoid gel (DES 10 -FLy 30 -GA 30) Add 25μL, 50μL and 100μL to 5mL sample bottles, add 1mL of diluted Escherichia coli solution to each of the three sample bottles as experimental group 1, experimental group 2 and experimental group 3; take an empty sample bottle and add 1mL of diluted Escherichia coli solution as the control group. Put the above 4 sample bottles in a 37℃ incubator and incubate for 24h; take four agar plates and spread the above four groups of incubated bacterial solutions respectively. Specifically, divide each agar plate into three equal parts for parallel samples, add 15μL of incubated bacterial solution to each part, and then use a disposable spreading stick to spread it evenly in this area, put the coated agar plate in a 37℃ incubator and incubate overnight. After incubation, place the agar plate under a colony counter to take pictures and observe the colony distribution of Escherichia coli.

[0070] The self-assembled eutectoid gel (DES 10 -FLy 30 -GA 30 )25μL, 50μL and 100μL were added to 5mL sample bottles, and 1mL of diluted Staphylococcus aureus solution was added to each of the three sample bottles as experimental group 1, experimental group 2 and experimental group 3; an empty sample bottle was added with 1mL of diluted Staphylococcus aureus solution as the control group. The above 4 sample bottles were placed in a 37°C incubator for 24 hours; four agar plates were taken and coated with the above four groups of incubated bacterial solutions respectively. Specifically, each agar plate was divided into three equal parts for parallel samples, 15μL of incubated bacterial solution was added to each part, and then it was evenly spread in this area with a disposable coating stick, and the coated agar plate was placed in a 37°C incubator for overnight incubation. After the incubation was completed, the agar plate was placed under a colony counter for photographing to observe the colony distribution of Staphylococcus aureus.

[0071] Figure 3 Self-assembled eutectoid gel DES 10 -FLy 30 -GA 30 The colony statistics of Escherichia coli and Staphylococcus aureus. Figure 3 The upper middle part is a colony statistics chart of Escherichia coli, from left to right are the control sample, experimental group 1, experimental group 2 and experimental group 3. Figure 3 The lower middle part is a colony statistics chart of Staphylococcus aureus, from left to right are the control sample, experimental group 1, experimental group 2 and experimental group 3.

[0072] from Figure 3 It can be seen that the self-assembled eutectoid gel DES 10 -FLy 30 -GA 30 It has strong antibacterial effect on Escherichia coli and Staphylococcus aureus, and its bactericidal property increases with the increase of gel amount.

[0073] The self-assembled eutectoid gel DES prepared in Example 1 was 10 -FLy 30 -GA 30 , DES10-FLy30 prepared in Comparative Example 1, DES10-GA30 prepared in Comparative Example 2, and FLy30-GA30 prepared in Comparative Example 3 were subjected to macrophage flow cytometry test, specifically:

[0074] RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin in an environment of 95% air and 5% CO2 at 37°C. RAW264.7 cells were then inoculated in 6-well plates (10 6 The cells were cultured overnight and then treated with LPS (1 μg / mL) for 12 hours to polarize to the M1 phenotype.

[0075] DES 10 -FLy 30 -GA 30 、DES 10 -FLy 30 、DES 10 -GA 30 and FLy 30 -GA 30 Dispersed in DMEM medium at a concentration of 1 μL / mL, four groups of dispersions were prepared; purified using 0.45 μm and 0.22 μm syringe filters to achieve sterile conditions. Subsequently, the cells were cultured with the above four dispersions for 24 hours for flow cytometry. The cells were transferred from the well plate to a centrifuge tube with a cell scraper and collected by centrifugation (1500 rpm, 5 minutes). After co-culturing the cells with CD86 dye in the dark at 4°C for 30 minutes, the cells were washed with PBS buffer, fixed with 4% paraformaldehyde for 15 minutes, and treated with BD membrane permeabilizer (1:10 dilution) for 30 minutes. After rinsing with PBS, incubated with CD206 dye in a dark environment at 4°C for 30 minutes. After rinsing with PBS, the cells were resuspended in 1 mL PBS and placed in a flow cytometer. Intracellular red fluorescence was detected by the PE channel, and intracellular green fluorescence was detected by the APC channel.

[0076] Figure 4 The flow cytometry diagrams of cells in the control group and LPS group after treatment with four types of co-eutectoid gels. The control group is a group that is not treated with LPS and co-eutectoid gels; the LPS group is a group that is only treated with LPS and not treated with co-eutectoid gels. Figure 4The small figure in the figure shows the ratio of M1 phenotype and M2 phenotype of macrophages after different co-precipitation gel treatments, where the red box indicates the ratio of M1 pro-inflammatory phenotype cells, and the blue box indicates the ratio of M2 anti-inflammatory phenotype cells. The higher the red M1 ratio, the stronger the inflammation and the worse the treatment effect; the higher the blue M2 ratio, the lower the inflammation and the better the treatment effect.

[0077] Depend on Figure 4 It can be seen that when GA is present, the prepared eutectoid gel has better anti-inflammatory properties. When DES is not present in the components, the content of M2 phenotype macrophages is less than that of the group with DES as the active liquid component, indicating that DES can provide cell membrane permeability for the eutectoid gel, making it easier for the small molecule anti-inflammatory drug GA to enter the cell. DES prepared in Example 1 10 -FLy 30 -GA 30 Has the best effect.

[0078] Although the present application has been described in detail in general terms and in specific embodiments in this specification, it is obvious to those skilled in the art that some modifications or improvements may be made to the present application. Therefore, these modifications or improvements made without departing from the spirit of the present application are within the scope of protection claimed in the present application.

Claims

1. A method for preparing a self-assembled co-evolution gel with cell permeability and multiple anti-inflammatory effects, characterized in that: include: S1, mixing choline chloride and a hydroxyl type hydrogen bond donor and heating them to obtain a viscous liquid; dissolving the viscous liquid in water to prepare a DES solution; S2, adding antibacterial active ingredients and lysozyme fibers to the DES solution, heating and ultrasonic treatment, and standing to room temperature to obtain a self-assembled eutectoid gel.

2. The preparation method according to claim 1, characterized in that: The hydroxyl type hydrogen bond donor is mannose or xylitol.

3. The preparation method according to claim 1, characterized in that: The antibacterial active ingredient is gallic acid or epigallocatechin gallate.

4. The preparation method according to claim 1, characterized in that: The molar ratio of the choline chloride to the hydroxyl type hydrogen bond donor is (4:1) to (1:4).

5. The preparation method according to claim 1, characterized in that: The concentration of the DES solution is 5-90 wt %.

6. The preparation method according to claim 1, characterized in that: The amount of the antibacterial active ingredient added to the DES solution is 10-50 mg / mL.

7. The preparation method according to claim 1, characterized in that: The amount of the lysozyme fiber added to the DES solution is 10-50 mg / mL.

8. The preparation method according to claim 1, characterized in that: In S1, the heating temperature is 50 to 90°C; In S2, the heating temperature is 30 to 50°C.

9. The self-assembled co-evolution gel having cell permeability and multiple anti-inflammatory effects prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the self-assembled eutectoid gel according to claim 9 in drugs for treating inflammatory diseases.

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