Injectable composite hydrogel as well as preparation method and application thereof

Abstract

The invention discloses injectable composite hydrogel as well as a preparation method and application thereof. The preparation method comprises the following steps: preparing freeze-dried silk fibroin into a silk fibroin aqueous solution; the preparation method comprises the following steps: dissolving polyoxyethylene polyoxypropylene ether powder in deionized water to obtain a polyoxyethylene polyoxypropylene ether aqueous solution; uniformly mixing the silk fibroin aqueous solution with the polyoxyethylene polyoxypropylene ether aqueous solution to obtain a silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution; and adding graphene oxide quantum dot powder into the silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution, uniformly stirring, and standing to obtain the injectable composite hydrogel. The injectable composite hydrogel has good biocompatibility, photo-thermal performance and angiogenic activity, can effectively resist bacteria on skin infected wounds under near-infrared laser irradiation, is a non-antibiotic dependent antibacterial treatment method, and effectively avoids the problem of drug resistance caused by antibiotic treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to an injectable composite hydrogel, a preparation method and application thereof. Background Art

[0002] Skin wound infection is a common clinical problem, usually caused by microbial infection. Its symptoms include redness, swelling, pain, pus exudation and fever, which seriously troubles the patient's recovery and quality of life. At present, the treatment for skin wound infection is mainly antibiotic treatment and other auxiliary treatments such as bandages and gauze. Antibiotic therapy has application limitations to a certain extent, such as low bioavailability, drug resistance and inappropriate treatment effect. Secondly, some infected wounds may be caused by a variety of pathogenic microorganisms, requiring different types of antibiotics, which increases the complexity of treatment. Auxiliary treatments such as bandages and gauze have no obvious antibacterial and repair-promoting abilities. They are difficult to operate when applied to deep infections and lacunar infections, and the clinical treatment effect is limited. Infected wounds not only prolong the healing time, but also increase the use of medical resources and treatment costs. In addition, the widely used antibiotics in the treatment of infected wounds have also caused drug resistance problems, posing a threat to public health. Summary of the invention

[0003] The purpose of the present invention is to provide an antibiotic-free antibacterial dressing which has good biocompatibility and strong antibacterial effect and can promote skin wound infection.

[0004] In order to achieve the above object, the present invention provides a method for preparing an injectable composite hydrogel, the preparation method comprising the following steps:

[0005] S1, preparation of silk fibroin aqueous solution:

[0006] preparing a silk fibroin aqueous solution by preparing the freeze-dried silk fibroin;

[0007] S2, preparation of polyoxyethylene polyoxypropylene ether aqueous solution:

[0008] dissolving polyoxyethylene polyoxypropylene ether powder in deionized water to obtain a polyoxyethylene polyoxypropylene ether aqueous solution;

[0009] S3, preparation of silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution:

[0010] The silk fibroin aqueous solution and the polyoxyethylene polyoxypropylene ether aqueous solution are uniformly mixed to obtain a silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution;

[0011] S4, Preparation of injectable composite hydrogels:

[0012] The graphene oxide quantum dot powder is added into the silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution, stirred evenly and then allowed to stand to obtain an injectable composite hydrogel.

[0013] Optionally, in S1, the method for preparing freeze-dried silk fibroin comprises the following steps:

[0014] Step 1, placing the silk cocoons in a Na2CO3 solution for water bath degumming to obtain degummed silk, and drying the silk;

[0015] Step 2, mixing calcium chloride, water and ethanol to obtain a ternary solution, heating the ternary solution to 100° C., adding the silk thereto, stirring until the silk is dissolved, and centrifuging at room temperature;

[0016] Step 3, dialyzing the supernatant after centrifugation, and freeze-drying the dialyzed solution to obtain freeze-dried silk fibroin.

[0017] Optionally, in S1, the mass concentration of the silk fibroin aqueous solution is 3% to 6%.

[0018] Optionally, in S2, the mass concentration of the polyoxyethylene polyoxypropylene ether aqueous solution is 20% to 40%.

[0019] Optionally, in S3, the volume ratio of the silk fibroin aqueous solution to the polyoxyethylene polyoxypropylene ether aqueous solution is (1-5):5.

[0020] Optionally, in S4, the mass concentration of the graphene oxide quantum dots is 50 μg / mL to 100 μg / mL.

[0021] The present invention also provides an injectable composite hydrogel, which is prepared by any one of the above-mentioned preparation methods.

[0022] Optionally, the injectable composite hydrogel increases in temperature under near-infrared laser irradiation.

[0023] Optionally, the wavelength of the near-infrared laser is 808 nm.

[0024] The present invention also provides an application of an injectable composite hydrogel, and the application of the injectable composite hydrogel in the repair and regeneration of skin infection wounds.

[0025] Compared with the prior art, the beneficial effects of the present invention include at least:

[0026] (1) The present invention compounds silk fibroin, polyoxyethylene polyoxypropylene ether, and graphene oxide quantum dots to prepare an injectable composite hydrogel antibacterial dressing of silk fibroin / polyoxyethylene polyoxypropylene ether / graphene oxide quantum dots. As the temperature rises, the polyoxyethylene polyoxypropylene ether can change from a liquid state to a solid state, thereby gradually dissolving from water and forming micron-scale hydrophobic domains through hydrophobic action. These hydrophobic domains provide physical support points for silk fibroin and promote the formation of a β-folding structure of silk fibroin. At the same time, the hydrogen bonds between silk fibroin molecules and the interaction between the hydrophobic domains intensify, resulting in the self-assembly of silk fibroin into a highly ordered β-folding structure. These β-folding structures are further cross-linked in the microscopic network structure of polyoxyethylene polyoxypropylene ether to form a stable composite hydrogel network. The injectable composite hydrogel antibacterial dressing has good biocompatibility and photothermal properties, has good angiogenic activity, can provide a favorable moist environment for tissue repair, and under the irradiation of near-infrared laser, can effectively carry out antibacterial treatment based on the photothermal effect of polyoxyethylene polyoxypropylene ether, providing a new type of non-antibiotic-dependent antibacterial treatment method for skin infection wounds, avoiding the generation of drug resistance.

[0027] (2) The injectable composite hydrogel of the present invention is in an injectable state and can be filled in situ into a skin infection wound under the action of external squeezing stimulation, and can self-repair to fit irregular skin infection wound defects. It has strong operability and is not limited by the complex shape of the skin infection wound. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the preparation method of the injectable composite hydrogel of the present invention.

[0029] Figure 2 This is a photothermal performance diagram of the injectable composite hydrogel of the present invention:

[0030] A is a photothermal imaging comparison diagram of the injectable composite hydrogel of the present invention and the control group; B is a comparison diagram of the temperature change over time of the injectable composite hydrogel of the present invention and the control group under laser irradiation.

[0031] Figure 3 This is a graph showing the effect of the injectable composite hydrogel of the present invention on cell proliferation.

[0032] Figure 4 The results of the injectable composite hydrogel of the present invention on cell migration and angiogenesis are shown in the figure below:

[0033] A is a comparison diagram of the effects of the injectable composite hydrogel of the present invention and the control group on cell migration; B is a comparison diagram of the effects of the injectable composite hydrogel of the present invention and the control group on angiogenesis. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0035] The experimental methods not mentioned in the present invention are all conventional experimental methods in the art, and the experimental materials not mentioned in the present invention are all conventionally commercially available.

[0036] At present, the treatment for skin infection wounds is mainly bandages, gauze and antibiotics. Bandage and gauze treatment lacks antibacterial effect and cannot kill or inhibit pathogens of wound infection; it cannot control humidity and it is difficult to maintain a moist environment around the wound, which is not conducive to promoting wound healing. Frequent replacement is required, which brings inconvenience to patients and may interfere with wound healing. If antibiotic treatment is used for a long time or inappropriately, it is easy for pathogens to develop tolerance to drugs, causing the originally effective antibiotics to gradually lose their efficacy. This is a key problem that needs to be solved in the current medical field. Hydrogel materials have a 3D spatial structure and can provide a moist environment for wound repair, making them a good skin dressing. However, the hydrogel skin dressings currently used in clinical practice have insufficient antibacterial properties, are easy to breed bacteria, and lack relevant biological activity (such as vasculogenic activity), which is difficult to meet the complex environment of clinical infectious wound repair. Therefore, it is urgent to develop new injectable skin dressings that have both antibiotic-independent antibacterial properties and vasculogenic activity.

[0037] Hydrogel materials can not only provide mechanical support for tissue repair, but also maintain a moist environment that is conducive to wound healing. They are considered to be ideal scaffold materials in tissue engineering. The present invention combines the advantages of silk fibroin (SF) hydrogel and polyoxyethylene polyoxypropylene ether (F-127) hydrogel, introduces the special catalytic activity and photothermal properties of graphene oxide quantum dots (GOQDs), and prepares a natural polymer injectable composite hydrogel antibacterial dressing. Among them, silk fibroin hydrogel can reduce (buffer) the biotoxicity of the other two materials, thereby improving the biocompatibility of the injectable composite hydrogel. The introduction of graphene oxide quantum dots can, on the one hand, act as a photothermal agent to produce a thermal effect, and on the other hand, can increase the overall hydrophilicity with the help of its rich oxygen-containing groups, thereby increasing biocompatibility.

[0038] The injectable composite hydrogel antibacterial dressing has good biocompatibility and photothermal properties, good angiogenic activity, and can provide a favorable moist environment for tissue repair. Under near-infrared laser irradiation, the photothermal effect of the material can be used to convert light energy into heat energy, thereby generating local high temperature, leading to bacterial cell membrane rupture, protein denaturation and DNA damage, thereby achieving effective antibacterial. Photothermal antibacterial therapy can show antibacterial activity against a variety of pathogens, including antibiotic-resistant bacteria and bacteria in biofilms; compared with traditional antibiotic treatment, photothermal antibacterial treatment usually only takes a few minutes to achieve better treatment effects; by controlling the intensity and time of the light source, the temperature of the treatment area can be precisely controlled to reduce damage to surrounding normal tissues. It provides a new type of non-antibiotic-dependent antibacterial treatment for skin infection wound repair.

[0039] Example

[0040] This embodiment provides a method for preparing an injectable composite hydrogel, comprising the following steps:

[0041] Step 1, preparation of freeze-dried silk fibroin:

[0042] (1) placing the silk cocoons in a 0.5 wt % Na2CO3 solution and degumming them in a 100° C. water bath for 30 minutes, repeating the degumming three times to obtain degummed silk;

[0043] (2) drying the degummed silk at 60°C for 6 hours;

[0044] (3) preparing a ternary solution of calcium chloride, water and ethanol at a molar ratio of 1:8:2, heating the ternary solution to 100° C., weighing the dried silk, and adding the dried silk to the ternary solution at a mass ratio of 1:25, stirring until the silk is completely dissolved, and centrifuging at room temperature to remove insoluble impurities;

[0045] (4) The supernatant after centrifugation was placed in a 3500 molecular weight dialysis bag and dialyzed under distilled water for 2 days;

[0046] (5) The dialysis solution is freeze-dried to obtain freeze-dried silk fibroin.

[0047] Step 2, preparation of silk fibroin aqueous solution:

[0048] A certain amount of freeze-dried silk fibroin was taken to prepare a silk fibroin aqueous solution with a final concentration of 5% (wt / v).

[0049] Step 3, preparation of F-127 aqueous solution:

[0050] A certain amount of F-127 powder was taken and dissolved in deionized water to prepare an F-127 aqueous solution with a final concentration of 30% (wt / v).

[0051] Step 4, preparation of SF / F-127 mixed solution:

[0052] The silk fibroin aqueous solution and the F-127 aqueous solution were mixed evenly according to a volume ratio of (1-5):5 to obtain a SF / F-127 mixed solution.

[0053] Step 5, preparation of injectable composite hydrogel:

[0054] Different masses of graphene oxide quantum dot powder were added to the above SF / F-127 mixed solution and stirred evenly at 4°C to obtain injectable GOQDs / SF / F-127 composite hydrogels with concentrations of 50 μg / mL to 100 μg / mL.

[0055] 1. Determination of photothermal properties of injectable composite hydrogels:

[0056] Deionized water (control group) and the injectable composite hydrogel prepared by the present invention were irradiated by near-infrared laser, and the parameters of the near-infrared laser were set to 808 nm, 5 min, 2 W / cm 2 The photothermal properties of the injectable composite hydrogel under 808 nm laser irradiation were recorded using a thermal imager. The specific experimental method is as follows:

[0057] S1: Prepare 200 μL EP tubes, pipette tips, pipettes, deionized water, injectable composite hydrogel samples, and photothermal imagers, and place them in the appropriate positions in order;

[0058] S2: Add deionized water and injectable composite hydrogel samples into the EP tubes respectively;

[0059] S3: Irradiation with near-infrared laser (808 nm, 5 min, 2 W / cm 2 ), and simultaneously record the temperature changes with a photothermal imager.

[0060] Experimental results, such as Figure 2 As shown, the results show that the injectable composite hydrogel has good photothermal properties. The temperature can reach 50°C after 5 minutes of laser irradiation, which is an ideal temperature for exerting photothermal antibacterial effects.

[0061] 2. Effects of injectable composite hydrogels on cell proliferation:

[0062] The injectable composite hydrogel prepared by the present invention was co-cultured with mesenchymal stem cells (key cells for tissue repair) for 1, 3, and 5 days to study its effect on the proliferation of mesenchymal stem cells. The specific experimental method is as follows:

[0063] S1: Prepare pipette tips, pipettes, trypsin, DMEM culture medium, 24-well plates and other consumables as well as well-grown mesenchymal stem cells;

[0064] S2: After digestion, the mesenchymal stem cells are seeded into a 24-well plate and allowed to grow to a density that accounts for 80% of the plate area;

[0065] S3: Scratch the middle of the 24-well plate with a 200 μL pipette tip and wash away the floating cells with PBS solution;

[0066] S4: A Transwell plate with injectable composite hydrogels was added to the top layer of a 24-well plate for co-culture;

[0067] S5: Samples were taken at 0, 12, and 24 h, and immunofluorescence staining was performed and the scratch area was counted.

[0068] The control group received routine treatment without any special intervention.

[0069] Experimental results: Figure 3 As shown, this indicates that the injectable composite hydrogel can significantly promote the proliferation of mesenchymal stem cells.

[0070] 3. Effects of injectable composite hydrogels on endothelial cell migration and angiogenesis:

[0071] The injectable composite hydrogel prepared by the present invention was co-cultured with vascular endothelial cells to observe its effects on vascular endothelial migration and angiogenesis. The specific experimental method is as follows:

[0072] S1: Prepare 24-well plates, pipette tips, pipette guns, matrix gel and other consumables and sterilize them with UV light in advance;

[0073] S2: Spread the matrix gel evenly into a 24-well plate and place in a 4°C refrigerator overnight;

[0074] S3: The digested vascular endothelial cells were seeded into a 24-well plate, and a Transwell plate with injectable composite hydrogel was added to the upper layer of the experimental group for co-culture;

[0075] S4: Samples were taken after 6 hours and fluorescent staining was performed to observe the angiogenesis of endothelial cells.

[0076] Among them, the control group only had culture medium and no other materials were added.

[0077] Experimental results; such as Figure 4 As shown, the results showed that the injectable composite hydrogel can promote the migration of vascular endothelial cells and their differentiation into angiogenesis.

[0078] In summary, the present invention provides a method for preparing an injectable composite hydrogel, wherein freeze-dried silk fibroin is formulated into a silk fibroin aqueous solution, polyoxyethylene polyoxypropylene ether powder is dissolved in deionized water to obtain a polyoxyethylene polyoxypropylene ether aqueous solution, the silk fibroin aqueous solution is mixed with the polyoxyethylene polyoxypropylene ether aqueous solution to obtain a silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution, and finally graphene oxide quantum dot powder is added to the silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution to obtain an injectable composite hydrogel. The injectable composite hydrogel has good photothermal antibacterial properties and angiogenic activity, can be used for the treatment of skin infected wounds, solves the problem of bacterial resistance caused by long-term use of antibiotic therapy, and is a new type of non-antibiotic-dependent antibacterial therapeutic material.

[0079] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing an injectable composite hydrogel, characterized in that: The preparation method comprises the following steps: S1, preparation of silk fibroin aqueous solution: preparing a silk fibroin aqueous solution by preparing the freeze-dried silk fibroin; S2, preparation of polyoxyethylene polyoxypropylene ether aqueous solution: dissolving polyoxyethylene polyoxypropylene ether powder in deionized water to obtain a polyoxyethylene polyoxypropylene ether aqueous solution; S3, preparation of silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution: The silk fibroin aqueous solution and the polyoxyethylene polyoxypropylene ether aqueous solution are uniformly mixed to obtain a silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution; S4, Preparation of injectable composite hydrogels: The graphene oxide quantum dot powder is added into the silk fibroin / polyoxyethylene polyoxypropylene ether mixed solution, stirred evenly and then allowed to stand to obtain an injectable composite hydrogel.

2. The preparation method according to claim 1, characterized in that In S1, the preparation method of the freeze-dried silk fibroin comprises the following steps: Step 1, placing the silk cocoons in a Na2CO3 solution for water bath degumming to obtain degummed silk, and drying the silk; Step 2, mixing calcium chloride, water and ethanol to obtain a ternary solution, heating the ternary solution to 100° C., adding the silk thereto, stirring until the silk is dissolved, and centrifuging at room temperature; Step 3, dialyzing the supernatant after centrifugation, and freeze-drying the dialyzed solution to obtain freeze-dried silk fibroin.

3. The preparation method according to claim 1, characterized in that: In S1, the mass concentration of the silk fibroin aqueous solution is 3% to 6%.

4. The preparation method according to claim 1, characterized in that: In S2, the mass concentration of the polyoxyethylene polyoxypropylene ether aqueous solution is 20% to 40%.

5. The preparation method according to claim 1, characterized in that: In S3, the volume ratio of the silk fibroin aqueous solution to the polyoxyethylene polyoxypropylene ether aqueous solution is (1-5):

5.

6. The preparation method according to claim 1, characterized in that: In S4, the mass concentration of the graphene oxide quantum dots is 50 μg / mL to 100 μg / mL.

7. An injectable composite hydrogel, characterized in that: The injectable composite hydrogel is prepared by the preparation method described in any one of claims 1 to 6.

8. The injectable composite hydrogel according to claim 7, characterized in that The temperature of the injectable composite hydrogel rises under the irradiation of near-infrared laser.

9. The injectable composite hydrogel according to claim 8, characterized in that The wavelength of the near-infrared laser is 808 nm.

10. A use of the injectable composite hydrogel according to any one of claims 7 to 9, characterized in that: Application of the injectable composite hydrogel in the repair and regeneration of skin infected wounds.

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