MXene carbon quantum dot hydrogel, preparation method and application

Abstract

The invention relates to the field of biological medicine application, in particular to MXene carbon quantum dot hydrogel and a preparation method and application thereof.The MXene carbon quantum dot hydrogel comprises MXene, carbon quantum dots and methacrylic acid hydrogel, and after the methacrylic acid hydrogel (GelMA) is wrapped with the MXene and the carbon quantum dots (CDs), the MXene / CDs hydrogel is obtained. The MXene / carbon quantum dot hydrogel disclosed by the invention has good antibacterial and osteogenic properties, in-vitro experiments prove that MXene has good antibacterial properties, and CDs has good capability of promoting osteogenic differentiation of bone marrow mesenchymal stem cells; meanwhile, the MXene / CDs hydrogel can have antibacterial and osteogenesis promoting performance, and a new thought is provided for clinical treatment of infectious bone defects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical applications, and in particular to a MXene carbon quantum dot hydrogel, a preparation method and an application thereof. Background Art

[0002] In the field of orthopedics, the repair of infected bone defects has always been an extremely difficult clinical problem that has troubled surgeons, affecting up to one million patients worldwide each year, bringing heavy economic and social burdens. Infected bone defects refer to inflammatory diseases caused by pathogens infecting the bone marrow, cortical bone, and periosteum, often leading to bone destruction. Current treatment methods generally focus on surgical treatment and antibiotic treatment, but the treatment cycle is long, the cost is high, the recurrence rate is high, and the treatment is difficult, making it extremely difficult to handle.

[0003] The research and development of bone tissue repair materials provides a new means for the treatment of bone defects. At present, metal implants represented by titanium and ceramic implants such as calcium phosphate have been successfully used in orthopedic clinics. The above materials have excellent mechanical strength and good biocompatibility, but due to the lack of effective antibacterial ability, invading local pathogens can colonize on the surface of the implant and form a biofilm, which becomes a hotbed for bacterial reproduction. Clinically, secondary surgery to remove the implant, thorough debridement, drug-loaded bone cement filling, systemic use of antibiotics and other means are often used for anti-infection treatment, which brings a huge medical burden to patients. In order to break through the above difficulties, researchers are committed to developing new antibacterial osteogenic implants with high-efficiency antibacterial ability and excellent osteogenic activity.

[0004] Hydrogel scaffolds have excellent biocompatibility, degradability and structural controllability, which enable them to provide a good bioactive environment for cell proliferation and differentiation. However, hydrogels themselves cannot effectively fight bacteria and promote osteogenic differentiation. MXene is a transition metal carbide or nitride or carbonitride. As an emerging two-dimensional nanomaterial, it has an ultra-thin sheet structure and has great potential and good application prospects in terms of antibacterial properties. MXene can be sterilized by direct physical contact or by photothermal sterilization. Carbon quantum dots (CDs) have attracted widespread attention due to their bright multicolor luminescence, excellent biocompatibility, water dispersibility and simplicity of synthesis from different carbon sources. It has a wide range of potential biological uses, and its excellent ability to promote osteogenic differentiation of bone marrow mesenchymal stem cells has made it widely used in bone tissue repair. Therefore, preparing MXene and CDs into a composite with hydrogel can simultaneously have excellent properties of antibacterial, osteogenic and slowly degradable, providing a new idea for the treatment of infected bone defects.

[0005] In view of this, the present invention provides a MXene carbon quantum dot hydrogel, a preparation method and an application thereof. Summary of the invention

[0006] The purpose of the present invention is to provide a MXene carbon quantum dot hydrogel, a preparation method and an application thereof in view of the deficiencies in the prior art.

[0007] In order to solve the above technical problems, the following technical solutions are adopted: A MXene / carbon quantum dot hydrogel comprises MXene, carbon quantum dots and methacrylated hydrogel. After the MXene and carbon quantum dots are wrapped in the methacrylated hydrogel, a MXene / CDs hydrogel is obtained.

[0008] A further improvement based on the technical solution is that the MXene is Ti3C2 transition metal carbide.

[0009] The present invention proposes another technical solution: a method for preparing MXene / carbon quantum dot hydrogel, comprising the following steps: S1. Add 0.8-1.2 g of gelatin into 8-12 mL of PBS buffer and stir at 50-70° C. to dissolve the gelatin in the PBS buffer; then add 0.5-1.5 mL of methacrylic anhydride and stir vigorously at 35-60° C. for 2.5-3.5 h; then add preheated PBS into the above solution to terminate the reaction; then put the obtained solution into a dialysis bag and dialyze at 40-50° C. for one week; the dialyzed solution is freeze-dried to obtain a foamy hydrogel precursor; S2. Take 0.3-0.5 g of the foamy hydrogel precursor prepared above, and completely dissolve the foamy hydrogel precursor in a preheated 1-5 mL PBS solution to obtain a hydrogel precursor solution, then add 85-110 μL of MXene and 85-110 μL of CDs, and finally add 0.008-0.15 g of photosensitizer, and continue to irradiate under ultraviolet light until the hydrogel solidifies, and finally obtain MXene / CDs hydrogel.

[0010] A further improvement based on the technical solution is that in step S1, the dialysis bag is a 14000 Da dialysis bag.

[0011] A further improvement based on the technical solution is that in step S2, the concentration of MXene is 1 mg / mL.

[0012] A further improvement based on the technical solution is that in step S2, the concentration of CDs is 1 mg / mL.

[0013] A further improvement based on the technical solution is that in step S2, the continuous irradiation time under the ultraviolet lamp is 4-10 minutes.

[0014] The present invention proposes another technical solution: an in vitro antibacterial application of a MXene / carbon quantum dot hydrogel, taking the MXene / carbon quantum dot hydrogel prepared by the above method, using a flat plate coating method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material or using a live-dead staining method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material.

[0015] The present invention proposes another technical solution: an application of a MXene / carbon quantum dot hydrogel to promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro. The MXene / carbon quantum dot hydrogel prepared by the above method is first fixed in a 6-well plate and sterilized with ultraviolet light; then bone marrow mesenchymal stem cells from experimental mice are added to each well, and cultured continuously for 14 days, with the culture medium replaced every three days; finally, the bone marrow mesenchymal stem cells are treated and the staining results are photographed with an optical microscope.

[0016] The present invention proposes another technical solution: an application of MXene / carbon quantum dot hydrogel in in vitro safety verification, taking the MXene / carbon quantum dot hydrogel prepared by the above method, and using the live-dead staining method to evaluate the cytotoxicity of Xene / carbon quantum dot hydrogel to stem cells.

[0017] The above technical solution has the following beneficial effects: The present invention provides a MXene carbon quantum dot hydrogel, a preparation method and an application, and obtains a MXene / CDs hydrogel after encapsulating MXene and carbon quantum dots (CDs) into a methacrylate hydrogel (GelMA). The MXene / carbon quantum dot hydrogel of the present invention has good antibacterial and osteogenesis properties. In vitro experiments have shown that MXene has good antibacterial properties and CDs has good ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells; MXene / CDs hydrogel can also have antibacterial and osteogenesis properties, providing a new idea for the clinical treatment of infected bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The present invention will be further described below in conjunction with the accompanying drawings: Figure 1 Figure a is the morphology and structure of the hydrogel; Figure 1 Figure b is the morphology and structure diagram of MXene / CDs hydrogel.

[0019] Figure 2 Figure a shows the antibacterial effect of MXene / CDs hydrogel coated on a flat plate in vitro; Figure 2 Figure b shows the in vitro antibacterial live-dead staining effect of MXene / CDs hydrogel.

[0020] Figure 3 Middle a is the alizarin red staining effect of the control group stem cells in vitro osteogenic differentiation; Figure 3 Alizarin red staining effect of bMXene / CDs hydrogel in promoting osteodifferentiation in vitro.

[0021] Figure 4 The results of the control group and experimental group for in vitro safety verification of MXene / CDs hydrogel. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below through the accompanying drawings and embodiments. However, it should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the scope of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Example

[0023] See also Figure 1 A MXene / carbon quantum dot hydrogel comprises MXene, carbon quantum dots and methacrylated hydrogel, wherein the MXene and carbon quantum dots are encapsulated in the methacrylated hydrogel to obtain a MXene / CDs hydrogel. Specifically, the MXene is Ti3C2 transition metal carbide.

[0024] Based on the above MXene / carbon quantum dot hydrogel: a preparation method of MXene / carbon quantum dot hydrogel is proposed, comprising the following steps: S1. Add 0.8-1.2g gelatin to 8-12mL PBS buffer and stir at 50-70℃ to dissolve the gelatin in the PBS buffer; then add 0.5-1.5mL methacrylic anhydride and stir vigorously at 35-60℃ for 2.5-3.5h; then add preheated PBS to the above solution to terminate the reaction; then put the obtained solution into a dialysis bag, which is a 14000 Da dialysis bag. Dialyze at 40-50℃ for one week; the dialyzed solution is freeze-dried to finally obtain a foamy hydrogel precursor.

[0025] In the embodiment of the above step S1, preferably, gelatin is 1 g, PBS buffer is 10 mL, and methacrylic anhydride is 0.8 mL. The stirring temperature of gelatin added to PBS buffer is controlled to be 60°C, and the stirring temperature after adding methacrylic anhydride is controlled to be 50°C, and the stirring time is 3 hours. The content of preheated PBS is 40 mL, and the preheating temperature is 40°C.

[0026] S2. Take 0.3-0.5g of the foamy hydrogel precursor prepared above, and completely dissolve the foamy hydrogel precursor in a preheated 1-5 mL PBS solution to obtain a hydrogel precursor solution, then add 85-110μL of MXene and 85-110μL of CDs, the MXene concentration is 1 mg / mL, and the CDs concentration is 1 mg / mL. Finally, add 0.008-0.15g of photosensitizer, and continue to irradiate under ultraviolet light until the hydrogel solidifies. The continuous irradiation time under ultraviolet light is 4-10 minutes to finally obtain MXene / CDs hydrogel.

[0027] In the embodiment of the above step S2, preferably, 0.36 g of the foamy hydrogel precursor is used, the preheated PBS solution is 2 mL, the MXene and CDs content is 100 μL, the photosensitizer content is 0.01 g, and the continuous irradiation time under the ultraviolet lamp is generally 5 minutes.

[0028] Depend on Figure 1 A and Figure 1 From b, we can see that Figure 1 Middle a shows the morphology of the hydrogel, showing a typical porous sponge-like structure; Figure 1 Figure b shows the morphology of the MXene / CDs hydrogel. It can be observed that after freeze-drying, MXene / CDs agglomerates appear in the pores and there are many wrinkles on its surface. These features indicate that the modification of MXene / CDs increases the roughness of the hydrogel surface, which is conducive to the proliferation and adsorption of cells. Example

[0029] See also Figure 2 , an in vitro antibacterial application of MXene / carbon quantum dot hydrogel, taking the MXene / carbon quantum dot hydrogel prepared by the above method, using a flat plate coating method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material or using a live-dead staining method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material.

[0030] (1) First, the antibacterial activity of the material was tested using the plate coating method. MXene / CDs hydrogel was first prepared in a 96-well plate. The specific operation was to take 100 μL of the mixed solution of the hydrogel precursor solution with the material and photosensitizer added to the 96-well plate, and then place the 96-well plate under ultraviolet light for 5 minutes to solidify the MXene / CDs hydrogel. Next, 10 μL of bacterial solution with a concentration of 106 CFU / mL was carefully added to the surface of the hydrogel, and then placed it in a 37°C environment for 24 h. After the incubation, 1 mL of sterilized PBS was added to each well, and the surviving bacteria were suspended by gently shaking. Then, 200 μL of the suspension was accurately taken from each well, inoculated onto the agarose solid culture medium, and placed again at 37°C for 12 h. Finally, the in vitro bactericidal activity of the MXene / CDs hydrogel was determined by carefully counting the number of bacterial colonies growing on the agarose solid culture medium.

[0031] (2) The antibacterial activity of the material was tested using the live-dead staining method. The bacteria were inoculated on the MXene / CDs hydrogel composite material. After culturing for 3 days, the bacteria were stained using a live-dead staining kit. The specific operation was to add an appropriate amount of calcein-AM and propidium iodide (PI) to PBS to prepare a working solution. The working solution was then directly added to the culture system containing the bacteria and placed in a 37 °C incubator for 15 minutes. After the culture was completed, the stained bacteria were imaged and observed using a laser confocal microscope under excitation wavelengths of 490 nm and 545 nm, respectively. Before imaging, the sample needed to be carefully washed with PBS to remove excess dye, so as to obtain clear and accurate images for evaluating the antibacterial activity of MXene / CDs hydrogels.

[0032] Depend on Figure 2 It can be seen that MXene / CDs hydrogel has excellent antibacterial properties. Specifically, Figure 2 Figure a is the antibacterial effect of MXene / CDs hydrogel coated on a flat plate in vitro, through which we can intuitively see the inhibitory effect of MXene / CDs hydrogel on bacteria; Figure 2 Figure b is the in vitro antibacterial live and dead staining effect of MXene / CDs hydrogel. From the figure, live bacteria (green fluorescence, calcein-AM staining) and dead bacteria (red fluorescence, propidium iodide staining) can be clearly distinguished, further proving the antibacterial properties of MXene / CDs hydrogel. Example

[0033] See also Figure 3, an application of MXene / carbon quantum dot hydrogel to promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro, taking the MXene / carbon quantum dot hydrogel prepared by the above method, fixing the prepared MXene / CDs hydrogel in a 6-well plate, and sterilizing it with ultraviolet light for 30 minutes. After sterilization, 500,000 mouse-derived bone marrow mesenchymal stem cells were added to each well, and then placed in a suitable culture environment for continuous culture for 14 days. During the culture process, the culture medium was replaced every three days to ensure that the nutrients required for cell growth were sufficient. After 14 days of culture, in order to evaluate the osteogenic differentiation effect of bone marrow mesenchymal stem cells, a series of treatments were required for the cells. First, the cells were washed with PBS for three times to remove impurities such as the culture medium. Then, the cells were fixed at room temperature with 4% paraformaldehyde for 30 min. After fixation, the cells were washed again with PBS for three times, and then washed with deionized water for two times to remove excess salt on the cell surface. Finally, 1 mL of Alizarin Red solution was added to each well and incubated at room temperature for 3-5 min to allow Alizarin Red to bind to the calcium salt in the osteoblasts. After staining, the cells were washed three times with PBS to remove the unbound Alizarin Red. Finally, the stained cells were photographed using an optical microscope to evaluate the effect of MXene / CDs hydrogel on the osteogenic differentiation of bone marrow mesenchymal stem cells by observing the staining results.

[0034] Depend on Figure 3 It can be seen that MXene / CDs hydrogel has excellent ability to promote the osteogenic differentiation of bone marrow mesenchymal stem cells. Specifically, Figure 3 Middle a is the alizarin red staining effect of the control group stem cells in vitro osteogenic differentiation. Figure 3 Figure b is the Alizarin red staining effect of MXene / CDs hydrogel on osteogenic differentiation in vitro. By comparing the two figures, it can be clearly seen that MXene / CDs hydrogel has a significant promoting effect on the osteogenic differentiation of bone marrow mesenchymal stem cells. Example

[0035] See also Figure 4 , an application of MXene / carbon quantum dot hydrogel in in vitro safety verification, taking the MXene / carbon quantum dot hydrogel prepared by the above method, using the live-dead staining method to evaluate the cytotoxicity of Xene / carbon quantum dot hydrogel to stem cells.

[0036] Specifically, stem cells were inoculated on MXene / CDs hydrogel composites. After culturing for 14 days, the stem cells were stained using a live-dead staining kit. An appropriate amount of calcein-AM and propidium iodide (PI) were added to PBS to prepare a working solution. The working solution was then added directly to the culture system containing stem cells and placed in a 37 °C incubator for 15 minutes. After the culture, the stained stem cells were imaged and observed using a laser confocal microscope under excitation at wavelengths of 490 nm and 545 nm, respectively. Before imaging, the sample needed to be carefully washed with PBS to remove excess dye to obtain a clear and accurate image. The in vitro safety of MXene / CDs hydrogels was evaluated based on the ratio of live cells (calcein-AM positive staining) and dead cells (propidium iodide positive staining) in the image.

[0037] Depend on Figure 4 It can be seen that MXene / CDs hydrogel has good biosafety. Figure 4 The results of in vitro safety verification of MXene / CDs hydrogel were presented. The growth of stem cells on the hydrogel was observed by live-dead staining method, and the cytotoxicity of the hydrogel to stem cells was evaluated. The results showed that MXene / CDs hydrogel has good biosafety.

[0038] Through the above examples, MXene / CDs hydrogel was comprehensively prepared and performance tested. The results showed that the prepared MXene / CDs hydrogel had good antibacterial osteogenesis and biosafety, and had potential application value in the treatment of infected bone defects, providing a new idea and method for the clinical treatment of infected bone defects.

[0039] The above are only specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent replacements or modifications made based on the present invention to solve basically the same technical problems and achieve basically the same technical effects are all included in the protection scope of the present invention.

Claims

1. A MXene / carbon quantum dot hydrogel, characterized in that: It includes MXene, carbon quantum dots and methacrylated hydrogel. After wrapping MXene and carbon quantum dots into methacrylated hydrogel, MXene / CDs hydrogel is obtained.

2. A MXene / carbon quantum dot hydrogel according to claim 1, characterized in that: The MXene is Ti3C2 transition metal carbide.

3. A method for preparing a MXene / carbon quantum dot hydrogel as claimed in claim 1 or 2, characterized in that The following steps are involved: S1. Add 0.8-1.2 g of gelatin into 8-12 mL of PBS buffer and stir at 50-70° C. to dissolve the gelatin in the PBS buffer; then add 0.5-1.5 mL of methacrylic anhydride and stir vigorously at 35-60° C. for 2.5-3.5 h; then add preheated PBS into the above solution to terminate the reaction; then put the obtained solution into a dialysis bag and dialyze at 40-50° C. for one week; the dialyzed solution is freeze-dried to obtain a foamy hydrogel precursor; S2. Take 0.3-0.5 g of the foamy hydrogel precursor prepared above, and completely dissolve the foamy hydrogel precursor in a preheated 1-5 mL PBS solution to obtain a hydrogel precursor solution, then add 85-110 μL of MXene and 85-110 μL of CDs, and finally add 0.008-0.15 g of photosensitizer, and continue to irradiate under ultraviolet light until the hydrogel solidifies, and finally obtain MXene / CDs hydrogel.

4. The method for preparing a MXene / carbon quantum dot hydrogel according to claim 3, characterized in that: In the step S1, the dialysis bag is a 14000 Da dialysis bag.

5. The method for preparing a MXene / carbon quantum dot hydrogel according to claim 3, characterized in that: In the step S2, the MXene has a concentration of 1 mg / mL.

6. The method for preparing a MXene / carbon quantum dot hydrogel according to claim 3, characterized in that: In the step S2, the CDs has a concentration of 1 mg / mL.

7. The method for preparing a MXene / carbon quantum dot hydrogel according to claim 3, characterized in that: In step S2, the irradiation time under the ultraviolet lamp is 4-10 minutes.

8. An in vitro antibacterial application of MXene / carbon quantum dot hydrogel, characterized in that: Take the MXene / carbon quantum dot hydrogel prepared by the method of any one of claims 3-7, and use the flat plate coating method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material or use the live-dead staining method to test the antibacterial activity of the MXene / carbon quantum dot hydrogel material.

9. An application of MXene / carbon quantum dot hydrogel to promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro, characterized in that: Take the MXene / carbon quantum dot hydrogel prepared by the method of any one of claims 3 to 7, first fix the MXene / carbon quantum dot hydrogel in a 6-well plate and sterilize it with ultraviolet light; then add bone marrow mesenchymal stem cells from experimental mice to each well, culture them continuously for 14 days, and change the culture medium every three days; finally, treat the bone marrow mesenchymal stem cells and photograph the staining results with an optical microscope.

10. An application of MXene / carbon quantum dot hydrogel for in vitro safety verification, characterized in that: Take the MXene / carbon quantum dot hydrogel prepared by the method of any one of claims 3 to 7, and use the live-dead staining method to evaluate the cytotoxicity of the Xene / carbon quantum dot hydrogel to stem cells.

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