Fe3O4-MXene material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of oral clinic, and particularly discloses a Fe3O4-MXene material and a preparation method and application thereof.The material comprises lithium fluoride, carbon titanium aluminum, hydrochloric acid, ethylene glycol, ferric chloride hexahydrate, 1, 6-hexamethylenediamine and sodium acetate; the lithium fluoride, the aluminum titanium carbide and the hydrochloric acid are used for synthesizing MXene, and the MXene, the ferric chloride hexahydrate, the 1, 6-hexamethylenediamine, the sodium acetate and the ethylene glycol are used for synthesizing Fe3O4-MXene; the invention aims to synthesize a novel root canal disinfection material with antibacterial and bone resorption inhibiting functions, the novel root canal disinfection material is Fe3O4-MXene, the Fe3O4-MXene is antibacterial through a photothermal effect, and Fe3O4 has superparamagnetism and can directionally move under the guide of a magnetic field, so that Fe3O4-MXene not only plays a role in a root canal, but also can enter an apical region to inhibit alveolar bone resorption; therefore, the primary target and the ultimate target of root canal therapy are promoted to be realized.

Description

Technical Field

[0001] The present invention relates to the field of oral clinical technology, and specifically to an Fe3O4-MXene material, a preparation method thereof, and an application thereof. Background Art

[0002] Periapical lesions that still do not heal after multiple standard root canal treatments are called refractory periapical periodontitis. Enterococcus faecalis is a pathogenic bacterium often isolated from reinfected root canals. Microorganisms in the root canal and the host inflammatory response jointly induce the formation of osteoclasts, ultimately leading to alveolar bone resorption. Therefore, the primary goal of root canal treatment for refractory periapical periodontitis is to control infection, and the ultimate goal is to promote the healing of periapical lesions.

[0003] Root canal treatment is the main treatment method for refractory periapical periodontitis. Chemical disinfection is one of the basic steps of root canal treatment. An ideal root canal disinfectant should simultaneously meet the dual goals of root canal treatment. Commonly used clinical chemical disinfectants such as sodium hypochlorite (NaClO) lack the function of inhibiting bone resorption. Currently, the research on new disinfection drugs mainly focuses on their antibacterial function, while ignoring the regulation of the host immune response. Therefore, it is necessary to explore a new type of root canal disinfectant that can both control infection and inhibit alveolar bone resorption to achieve the dual goals of root canal treatment.

[0004] In summary, it can be known that: traditional root canal disinfection drugs do not have the function of inhibiting bone resorption itself and have relatively high cytotoxicity; moreover, traditional root canal disinfection drugs cannot enter the apical region. Although traditional root canal disinfection drugs have good antibacterial effects, they cannot inhibit periapical bone resorption. For this reason, an Fe3O4-MXene material, a preparation method thereof, and an application thereof are provided. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies of the prior art and provide an Fe3O4-MXene material, a preparation method thereof, and an application thereof to solve the problems raised in the above background art.

[0006] To achieve the above purpose, the present invention provides the following technical solution: An Fe3O4-MXene material, which includes lithium fluoride, aluminum carbotitanide, hydrochloric acid, ethylene glycol, ferric trichloride hexahydrate, 1,6-hexanediamine, and sodium acetate; the lithium fluoride, aluminum carbotitanide, and hydrochloric acid are used to synthesize MXene, and the MXene, ferric trichloride hexahydrate, 1,6-hexanediamine, sodium acetate, and ethylene glycol are used to synthesize Fe3O4-MXene; Lithium fluoride: Aluminum carbotitanide: Hydrochloric acid = 8:8:57; MXene: Ferric trichloride hexahydrate: 1,6-Hexanediamine: Sodium acetate: Ethylene glycol = 9: 27: 97.2: 108: 6600.

[0007] A preparation method of the Fe3O4-MXene material as described above, the specific steps are as follows: Step 1: Synthesis of MXene: Dissolve 1 g of LiF and 1 g of Ti3AlC2 MAX phase in 20 mL of 9 M hydrochloric acid, stir magnetically at 35 °C for 24 hours; the reaction product is centrifuged at 3500 rpm and washed repeatedly with deionized water until pH≥6, and finally MXene is collected by centrifugation at the same speed and freeze-dried for standby; Step 2: Synthesis of Fe3O4-MXene: Disperse 90 mg of MXene in 60 mL of ethylene glycol and ultrasonically treat for 30 minutes, then add 0.27 g of FeCl3·6H2O, 0.972 g of 1,6-hexanediamine and 1.08 g of sodium acetate in sequence, stir for 1 hour and then transfer to a reaction kettle and react at 200 °C for 7 hours; the final product is magnetically separated and washed repeatedly with ethanol / deionized water, and dispersed in deionized water for use, thus obtaining the Fe3O4-MXene material.

[0008] An application of the Fe3O4-MXene material as described above in the preparation of a root canal disinfection material with photothermal antibacterial and bone resorption inhibition functions.

[0009] Compared with the prior art, the beneficial effects of the present invention are: The present invention synthesizes a novel root canal disinfection material Fe3O4-MXene with both antibacterial and bone resorption inhibition functions. Fe3O4-MXene antibacterial by photothermal effect, and Fe3O4 has superparamagnetism and can move directionally under the guidance of a magnetic field. Therefore, in addition to playing a role in the root canal, Fe3O4-MXene can also enter the apical region to inhibit alveolar bone resorption, thereby promoting the realization of the primary goal and ultimate goal of root canal treatment.

[0010] The present invention synthesizes a composite material, making full use of the respective advantages of MXene and Fe3O4, enabling Fe3O4-Mxene to play a role in photothermal antibacterial in the root canal and then enter the apical region to inhibit periapical bone resorption, and can play a role at two sites in the root canal and the apical region, overcoming the shortcomings of the existing root canal disinfection drugs with single function and ignoring the host function regulation in the periapical lesion area.

[0011] The advantage of the present invention is that it has both antibacterial and bone resorption inhibition functions. Because the present invention cleverly utilizes the characteristic of Fe3O4 moving directionally under a magnetic field to synthesize the bifunctional composite material Fe3O4-Mxene, and the respective characteristics of Fe3O4 and Mxene are not affected during the synthesis process, enabling Fe3O4-Mxene to enter the apical region to inhibit periapical bone resorption after playing a role in photothermal antibacterial in the root canal, and improving the curative effect of refractory periapical periodontitis at multiple sites. Description of the Drawings

[0012] Figure 1 is a flow chart of the method of the present invention; Figure 2 is a characterization diagram of Fe3O4-MXene of the present invention; Figure 3 is a photothermal performance diagram of Fe3O4-MXene of the present invention; Figure 4 is a biosafety diagram of Fe3O4-MXene of the present invention; Figure 5 is a photothermal antibacterial effect diagram of Fe3O4-MXene of the present invention; Figure 6 is a diagram of the inhibitory effect of Fe3O4-MXene of the present invention on bone resorption. Detailed implementation manners

[0013] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0014] Photothermal therapy (PTT) is a novel antibacterial method. PTT utilizes a photothermal agent to absorb near-infrared light between 700 - 950 nm, convert light energy into heat energy, gradually increase the temperature, cause the bacterial cell membrane to rupture and proteins to denature, and exert an antibacterial function. As a two-dimensional nanosheet photothermal material, MXene can absorb 808 nm near-infrared light and convert light energy into heat energy. Research has confirmed that MXene combined with 808 nm near-infrared light can effectively kill Enterococcus faecalis and other Gram-positive and Gram-negative bacteria. More importantly, MXene can inhibit the formation of osteoclasts. Thus, MXene can not only exert an efficient antibacterial effect through PTT but also inhibit the activity of osteoclasts, and is a highly potential PAP therapeutic agent.

[0015] To enable MXene to play an anti - bone - resorption role, it needs to reach the apical region. Previous studies have shown that under the action of an external magnetic field, the combination of Fe3O4 magnetic nanoparticles and traditional root canal sealants can increase the depth of the sealant entering the dentinal tubules. And Fe3O4 nanoparticles also have the function of inhibiting bone resorption. Therefore, combining Fe3O4 nanoparticles with MXene as a root canal disinfectant for PAP has good prospects: after MXene plays a photothermal antibacterial role in the root canal, Fe3O4 nanoparticles can transport MXene to the apical region under the guidance of a magnetic field to inhibit bone resorption, providing a root canal disinfection material with photothermal antibacterial and anti - bone - resorption functions. The root canal disinfection material Fe3O4 - MXene antibacterial by photothermal effect, and Fe3O4 has superparamagnetism and can move directionally under the guidance of a magnetic field. So Fe3O4 - MXene can not only play a role in the root canal, but also enter the apical region to inhibit alveolar bone resorption, thus promoting the realization of the primary and ultimate goals of root canal treatment.

[0016] Example 1: The synthesis method and characterization of Fe3O4 - MXene are as Figure 1 shown; Synthesis of MXene: MXene was synthesized by the reported method; the specific process is as follows: Dissolve 1 g of LiF and 1 g of Ti3AlC2 MAX phase in 20 mL of 9 M hydrochloric acid, and stir magnetically at 35 °C for 24 hours. The reaction product was centrifuged at 3500 rpm and washed repeatedly with deionized water until pH≥6, and finally MXene was collected by centrifugation at the same speed and freeze - dried for standby.

[0017] Synthesis of Fe3O4 nanoparticles: Fe3O4 nanoparticles were prepared by the solvothermal method. 0.69 g of FeCl3·6H2O, 0.24 g of trisodium citrate dihydrate and 1.98 g of ammonium acetate were successively added to a polytetrafluoroethylene - lined reaction kettle containing 36 mL of ethylene glycol, and stirred vigorously at room temperature for 1 hour. Subsequently, the reaction kettle was transferred to an oven at 200 °C for 16 hours. The product was collected by magnetic separation after being washed several times with ethanol and water.

[0018] Synthesis of Fe3O4 - MXene: Disperse 90 mg of MXene in 60 mL of ethylene glycol and ultrasonically treat for 30 minutes. Then add 0.27 g of FeCl3·6H2O, 0.972 g of 1,6 - hexanediamine and 1.08 g of sodium acetate in sequence, stir for 1 hour and then transfer to a reaction kettle at 200 °C for 7 hours. The final product was collected by magnetic separation and washed several times with ethanol / deionized water, and dispersed in deionized water for use.

[0019] Characterization of Fe3O4 - MXene: The crystal structure of Fe3O4-MXene was detected by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), vibrating sample magnetometer (VSM) and X-ray diffraction (XRD) patterns, and the surface morphology was detected by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As Figure 2 shown: (a) X-ray diffraction pattern (XRD): The characteristic peaks of MXene at 6.0° and 61.1°, and the standard diffraction peaks of Fe3O4 (JCPD 19-06290) of Fe3O4-MXene at 30.2° (200), 35.6° (311) and 56.2° (511) confirmed the crystal structure integrity of the composite material. (b) Fourier transform infrared spectroscopy (FTIR): The enhanced absorption peak of Fe-O bond at 560 cm −1 confirmed the successful modification of MXene by Fe3O4. (c-e) Scanning electron microscopy and (f-h) Transmission electron microscopy: MXene presented a typical two-dimensional layered wrinkled structure, while Fe3O4 nanoparticles in the composite material were evenly distributed on the surface of MXene. Clear Fe3O4 lattice fringes were visible in the high-resolution TEM, and the MXene structure remained intact, confirming the successful synthesis of the composite material. (i) Thermogravimetric analysis (TGA): The mass of Fe3O4 and Fe3O4-MXene decreased by 11.95% and 9.46% respectively, indicating that the composite material had good thermal stability. (j) Vibrating sample magnetometer (VSM): The magnetization intensity of Fe3O4-MXene was 25 emu / g and it moved under the attraction of a magnet within 10 seconds. (F: Fe3O4, M: MXene, FM: Fe3O4-MXene).

[0020] Example 2: Photothermal performance and biosafety evaluation of Fe3O4-MXene Photothermal performance First, irradiate 1 mL of Fe3O4-MXene solution with near-infrared light (5.7 W / cm²), and record the temperature change with an infrared thermal imager every minute. Subsequently, the absorbance of different concentrations of Fe3O4-MXene aqueous solution in the near-infrared region was measured by ultraviolet-visible spectrophotometer, and its photothermal performance was calculated and analyzed. In addition, five cycles of 10-minute light irradiation - 10-minute cooling cycle mode were carried out to evaluate the photothermal stability of Fe3O4-MXene under repeated light irradiation. As Figure 3Shown: (a - b) Temperature changes after illumination of Fe3O4-MXene at different concentrations: As the concentration of Fe3O4-MXene increased, the solution temperature increased by 23.4 to 39.3 degrees after 10 minutes of illumination. (c) Photothermal stability of Fe3O4-MXene: The photothermal performance of Fe3O4-MXene remained stable after 5 illumination-cooling cycles. (d) Visible-ultraviolet spectrum: As the concentration increased, the absorption of Fe3O4-MXene in the near-infrared light increased. (e) The extinction coefficient of Fe3O4-MXene at 808 nm was 1.12 L / (g·cm). (f) Temperature-time curve of 320 μg / mL Fe3O4-MXene from the highest temperature after illumination to natural cooling. (g) According to Figure 3 .f, the time constant τ = 351.9 s was calculated. Finally, through Figure 3 .e - g, the photothermal conversion efficiency of Fe3O4-MXene was calculated to be 45.67%. The above results indicate that Fe3O4-MXene has good photothermal performance.

[0021] Biosafety Mouse fibroblast L929 cells with good growth status and normal morphology were selected. After trypsin digestion, the cells were centrifuged at 1000 rpm for 1 min to collect the cells. The cells were seeded in 96-well plates at a density of 5000 cells / well and cultured at 37 °C and 5% CO2 for 24 h to allow the cells to cover the bottom of the wells. After removing the original medium, 100 μL of medium containing different concentrations of Fe3O4-MXene was added to each well in the experimental group, and 100 μL of normal medium was added to each well in the control group. The cells were incubated at 37 °C and 5% CO2 for 24 h. Subsequently, a CCK-8 kit was used for cytotoxicity detection: The old medium was removed, and the cells were rinsed three times with PBS. 100 μL of medium and 10 μL of CCK-8 reagent were added to each well, and the cells were incubated in the dark at 37 °C and 5% CO2 for 1 h. An experimental group containing only medium and CCK-8 solution was set as the blank group to avoid the influence of the color of the test solution and the culture medium itself. The supernatant was aspirated and transferred to a new 96-well plate, and the absorbance (OD) at a wavelength of 450 nm was measured using a multi-functional enzyme-linked detector SpectraMax iD5.

[0022] Single-rooted maxillary premolars without caries, root resorption or fracture were selected and stored in 75% ethanol for 1 week. The crowns were transected at the enamel-cementum junction, leaving a root length of approximately 12 mm, and the working length was defined at 0.5 mm from the apex. All teeth were prepared to a #40 / 06 taper using ProTaper nickel-titanium rotary instruments and irrigated with 1.0% sodium hypochlorite. After root canal preparation, the samples were placed in 1.5 mL sterile centrifuge tubes containing 1 mL PBS and autoclaved at 121 °C for 20 minutes. Aqueous solutions of Fe3O4-MXene at 0, 160, and 320 μg / mL were injected into the root canals, respectively, and irradiated vertically with an 808 nm laser (5.7 W / cm²) for 10 minutes. The temperature changes at three sites outside the root canal (the canal orifice, the middle of the root, and the apex) were recorded using the metal probe of a thermometer. A temperature change of less than 10 °C on the outer surface of the root could avoid periodontal tissue damage. As Figure 4 shown: (a) Compared with the control group, Fe3O4-MXene at 20 - 640 μg / mL had no obvious cytotoxicity to L929 cells. (b) Fe3O4-MXene at a concentration of 320 μg / mL or less did not cause the temperature change on the outer surface of the root to exceed 10 °C after light irradiation, which could avoid heat damage to the periodontal tissue. Therefore, the highest concentration of Fe3O4-MXene in the subsequent experiments was 320 μg / mL.

[0023] Example 3: Detection of the photothermal antibacterial function of Fe3O4-MXene Cultivation of Enterococcus faecalis Prepare brain heart infusion broth (BHI) liquid medium: Dissolve 14.8 g of BHI powder in 400 mL of deionized water, sterilize by high-pressure steam, and store at room temperature after cooling; BHI agar solid medium: Dissolve 14.8 g of BHI powder and 6 g of agar powder in 400 mL of BHI deionized water, sterilize by high-pressure steam, and pour plates after cooling to 50 °C. BHI medium containing sugar: Dissolve 14.8 g of BHI powder and 4 g of sucrose in 400 mL of deionized water, sterilize by high-pressure steam for later use. Enterococcus faecalis was resuscitated on a BHI agar plate by the streaking method and cultured overnight at 37 °C in an anaerobic chamber (N2: 90%, CO2: 5%, H2: 5%). Use an inoculation loop to pick a single colony and inoculate it into 10 mL of BHI liquid medium, and culture overnight at 37 °C in an anaerobic chamber (N2: 90%, CO2: 5%, H2: 5%). Dilute the bacteria in the logarithmic growth phase to 1×10 8 CFU / mL for later use. When culturing Enterococcus faecalis biofilm, dilute the bacterial solution to 2×10 6 CFU / mL with BHI containing sugar, then transfer the bacterial solution to a 48-well plate, 500 μL per well, and culture for one week, changing the liquid every other day, or add the bacterial solution to the prepared root canals of extracted teeth and culture for 3 weeks, changing the liquid every other day.

[0024] Inhibitory effect on planktonic Enterococcus faecalis Mix the diluted Enterococcus faecalis bacterial solution with Fe3O4-MXene solutions of different concentrations and irradiate for 0, 5, and 10 minutes respectively. After irradiation, perform gradient dilution on Enterococcus faecalis, and finally pipette 100 μL of the bacterial solution and evenly spread it on a BHI solid plate, and culture overnight and count in an anaerobic chamber (N2: 90%, CO2: 5%, H2: 5%) at 37°C.

[0025] Inhibitory effect on Enterococcus faecalis biofilm Remove the old culture medium and treat the Enterococcus faecalis biofilm according to the following groups: (1) PBS, (2) PBS + NIR irradiation, (3) Fe3O4-MXene, (4) Fe3O4-MXene + NIR irradiation, (5) 1% NaClO, (6) Fe3O4-MXene + NIR irradiation + 1% NaClO. The treatment method of NaClO is as follows: Let NaClO act for 30 seconds, and then add an equal volume of 5% mass fraction of sodium thiosulfate to terminate the reaction of NaClO. After the treatment, remove the supernatant, add BHI medium to scrape the Enterococcus faecalis at the bottom of the well plate for gradient dilution and plating. Or use a 40# file to collect the debris in the root canal of the extracted tooth, transfer the debris to an ep tube containing BHI medium, and perform gradient dilution and plating. As Figure 5 shown: (a) Inhibitory effect of Fe3O4-MXene on planktonic Enterococcus faecalis. Only when irradiated with 320 μg / mL Fe3O4-MXene for 10 minutes has a significant inhibitory effect. Therefore, the light irradiation conditions for subsequent experiments are 320 μg / mL and irradiation for 10 minutes. (b-c) Inhibitory effect of Fe3O4-MXene on Enterococcus faecalis biofilm in (b) the well plate and (c) the root canal of the extracted tooth: Photothermal therapy mediated by Fe3O4-MXene can effectively inhibit Enterococcus faecalis biofilm and can further enhance the antibacterial effect of the traditional root canal disinfection drug NaClO.

[0026] Example 4: Detection of the bone resorption inhibitory function of Fe3O4-MXene Osteoclast induction of RAW264.7 cells: Use α-MEM medium containing RANKL (100 ng / ml) to induce osteoclasts in RAW264.7 cells in a 48-well plate. Seed 1×10 4 RAW264.7 cells per well, change the medium on the 3rd day (100 ng / ml RANKL), and osteoclasts start to form on the 4th day.

[0027] Primary osteoclast induction: The long bones of 4-week-old C57 mice were taken, and bone marrow red blood cells were removed using red blood cell lysis buffer. After centrifugation, the red blood cell lysis buffer was removed, and the cells were resuspended in α-MEM medium containing 100 ng / ml MCSF and cultured overnight in a 6-cm cell culture dish treated with TC; then the non-adherent suspended cells were collected and continued to be cultured in a 6-cm cell culture dish treated with TC; after 48 h, the supernatant was discarded, and the adherent cells were collected and cultured in α-MEM medium containing 50 ng / ml MCSF and 50 ng / ml RANKL, with 5×10 4 cells per well, and the induction medium (50 ng / ml MCSF, 50 ng / ml RANKL) was changed every other day; osteoclasts could be obtained after 6-7 days.

[0028] Fe3O4-MXene treatment: Different concentrations of Fe3O4-MXene were co-cultured with RAW264.7 cells for osteoclast induction. The differentiation of osteoclasts was detected using a tartrate-resistant acid phosphatase (TRAP) staining kit. As Figure 6 shown: (a) The main constituent elements (C, O, Ti, Fe) of Fe3O4-MXene were detected on the slide at the apical side of the extracted tooth root, demonstrating that Fe3O4-MXene could reach the apical region through the apical foramen under the action of a magnetic field. (b-c) The inhibitory effects of different concentrations of Fe3O4-MXene on osteoclasts derived from the RAW264.7 cell line. 40 μg / mL Fe3O4-MXene could significantly inhibit osteoclast formation, indicating that only a small amount of the material reaching the apical region was needed to inhibit bone resorption. (c) The inhibitory effect of 40 μg / mL Fe3O4-MXene on primary osteoclasts.

[0029] The above embodiments only represent the implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. A Fe3O4-MXene material, characterized in that: The material comprises lithium fluoride, aluminum titanium carbide, hydrochloric acid, ethylene glycol, ferric chloride hexahydrate, 1,6 - hexanediamine and sodium acetate; the lithium fluoride, aluminum titanium carbide and hydrochloric acid are used for synthesizing MXene, and the MXene, ferric chloride hexahydrate, 1,6 - hexanediamine, sodium acetate and ethylene glycol are used for synthesizing Fe3O4 - MXene; Lithium fluoride: aluminum titanium carbide: hydrochloric acid = 8:8:57; MXene: ferric chloride hexahydrate: 1,6 - hexanediamine: sodium acetate: ethylene glycol = 9: 27: 97.2: 108: 6600.

2. A preparation method of the Fe3O4-MXene material as described in claim 1, characterized in that: The specific steps are as follows: Step 1: Synthesis of MXene: Dissolve 1 g of LiF and 1 g of Ti3AlC2 MAX phase in 20 mL of 9 M hydrochloric acid, and stir magnetically at 35 °C for 24 hours; the reaction product is centrifuged at 3500 rpm and washed repeatedly with deionized water until pH≥6, and finally MXene is collected by centrifugation at the same speed and freeze - dried for standby; Step 2: Synthesis of Fe3O4 - MXene: Disperse 90 mg of MXene in 60 mL of ethylene glycol and ultrasonically treat for 30 minutes, then successively add 0.27 g of FeCl3·6H2O, 0.972 g of 1,6 - hexanediamine and 1.08 g of sodium acetate, stir for 1 hour and then transfer to a reaction kettle and react at 200 °C for 7 hours; the final product is magnetically separated and washed repeatedly with ethanol / deionized water, and dispersed in deionized water for use, thus obtaining the Fe3O4 - MXene material.

3. Use of an Fe3O4 - MXene material as described in claim 1 in the preparation of a root canal disinfection material with photothermal antibacterial and bone resorption inhibition properties.

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