A Fe3O4-MXene material and its preparation method and application

Abstract

The present invention relates to the field of oral clinical technology, and specifically discloses an Fe3O4-MXene material, a preparation method thereof, and an application thereof. The material comprises lithium fluoride, aluminum carbotitanium, hydrochloric acid, ethylene glycol, ferric chloride hexahydrate, 1,6-hexanediamine, and sodium acetate. The lithium fluoride, aluminum carbotitanium, and hydrochloric acid are used to synthesize MXene, and the MXene, ferric chloride hexahydrate, 1,6-hexanediamine, sodium acetate, and ethylene glycol are used to synthesize Fe3O4-MXene. The present invention aims to synthesize a novel root canal disinfection material, Fe3O4-MXene, which has both antibacterial and bone absorption inhibition functions. Fe3O4-MXene is antibacterial through a photothermal effect, and Fe3O4 has superparamagnetism and can move in a direction under the guidance of a magnetic field. Therefore, in addition to playing a role in the root canal, Fe3O4-MXene can also enter the root apex to inhibit alveolar bone absorption, thereby promoting the realization of the primary and ultimate goals of root canal treatment.

Description

Technical Field

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

[0002] Periapical lesions that persist despite multiple standard root canal treatments are termed refractory apical periodontitis. Enterococcus faecalis is a common pathogen isolated from reinfected root canals. Root canal microorganisms, combined with the host's inflammatory response, induce osteoclast formation, ultimately leading to alveolar bone resorption. Therefore, the primary goal of root canal treatment for refractory apical periodontitis is to control infection, while the ultimate goal is to promote healing of the periapical lesion.

[0003] Root canal therapy (ROT) is the primary treatment for refractory apical periodontitis, and chemical disinfection is a fundamental step in ROT. An ideal ROT disinfectant should simultaneously achieve the dual goals of ROT. Commonly used chemical disinfectants, such as sodium hypochlorite (NaClO), lack the ability to inhibit bone resorption. Current research on novel disinfectants has primarily focused on their antimicrobial properties, while neglecting their potential to modulate the host immune response. Therefore, there is a need to explore novel ROT disinfectants that can both control infection and inhibit alveolar bone resorption, thereby achieving the dual goals of ROT.

[0004] In summary, traditional root canal disinfectants lack the ability to inhibit bone resorption and exhibit significant cytotoxicity. Furthermore, these agents are unable to penetrate the root apex. While they offer excellent antibacterial effects, they are unable to inhibit periapical bone resorption. To address this issue, an Fe3O4-MXene material, its preparation method, and its application are presented. Summary of the Invention

[0005] The purpose of the present invention is to provide a Fe3O4-MXene material and its preparation method and application in order to solve the problems raised by the above background technology in view of the defects of the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a Fe3O4-MXene material, which includes lithium fluoride, aluminum carbide, hydrochloric acid, ethylene glycol, ferric chloride hexahydrate, 1,6-hexanediamine, and sodium acetate; the lithium fluoride, aluminum carbide, and hydrochloric acid are used to synthesize MXene, and the MXene, ferric chloride hexahydrate, 1,6-hexanediamine, sodium acetate, and ethylene glycol are used to synthesize Fe3O4-MXene;

[0007] Lithium fluoride: titanium aluminum carbide: hydrochloric acid = 8:8:57; MXene: ferric chloride hexahydrate: 1,6-hexanediamine: sodium acetate: ethylene glycol = 9: 27: 97.2: 108: 6600.

[0008] A method for preparing the Fe3O4-MXene material as described above, the specific steps are as follows:

[0009] Step 1: Synthesis of MXene: 1 g of LiF and 1 g of Ti3AlC2 MAX were dissolved in 20 mL of 9 M hydrochloric acid and magnetically stirred at 35°C for 24 h. The reaction product was centrifuged at 3500 rpm and repeatedly washed with deionized water until the pH was ≥ 6. Finally, the MXene was collected by centrifugation at the same speed and freeze-dried for later use.

[0010] Step 2: Synthesis of Fe3O4-MXene: 90 mg of MXene was dispersed in 60 mL of ethylene glycol and ultrasonically treated for 30 minutes. 0.27 g of FeCl3·6H2O, 0.972 g of 1,6-hexanediamine, and 1.08 g of sodium acetate were added in sequence. After stirring for 1 hour, the mixture was transferred to a reactor and reacted at 200°C for 7 hours. The final product was magnetically separated and washed with ethanol / deionized water multiple times, and then dispersed in deionized water for use, thereby obtaining Fe3O4-MXene material.

[0011] 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 properties.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] The present invention synthesizes a new root canal disinfection material Fe3O4-MXene with both antibacterial and bone absorption inhibition functions. Fe3O4-MXene is antibacterial through the photothermal effect, and Fe3O4 has superparamagnetism and can move in a directed manner under the guidance of a magnetic field. Therefore, in addition to exerting its effect within the root canal, Fe3O4-MXene can also enter the apical area to inhibit alveolar bone absorption, thereby promoting the realization of the primary and ultimate goals of root canal treatment.

[0014] The present invention synthesizes a composite material that fully utilizes the respective advantages of MXene and Fe3O4, so that Fe3O4-MXene exerts a photothermal antibacterial effect in the root canal and then enters the root apex to inhibit periapical bone resorption. It can exert its effects at two sites, inside the root canal and in the root apex, overcoming the shortcomings of existing root canal disinfection drugs, which have a single function and ignore the host function regulation in the periapical lesion area.

[0015] The advantage of the present invention is that it has both antibacterial and bone resorption inhibiting functions, because the present invention cleverly utilizes the directional movement property of Fe3O4 under a magnetic field to synthesize the dual-functional composite material Fe3O4-Mxene, and the respective properties of Fe3O4 and Mxene are not affected during the synthesis process, so that Fe3O4-Mxene can enter the apical region after exerting its photothermal antibacterial effect in the root canal, inhibiting periapical bone resorption and improving the efficacy of refractory apical periodontitis at multiple sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a flow chart of the method of the present invention;

[0017] Figure 2 Characterization diagram of Fe3O4-MXene of the present invention;

[0018] Figure 3 This is the photothermal performance diagram of Fe3O4-MXene of the present invention;

[0019] Figure 4 This is a biosafety diagram of Fe3O4-MXene of the present invention;

[0020] Figure 5 This is a photothermal antibacterial effect diagram of Fe3O4-MXene of the present invention;

[0021] Figure 6 This is a diagram showing the bone resorption inhibition effect of Fe3O4-MXene of the present invention. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are 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 precise definition of the protection scope of the present invention.

[0023] Photothermal therapy (PTT) is a novel antibacterial approach. PTT utilizes a photothermal agent to absorb near-infrared light between 700 and 950 nm, converting the light energy into heat. This gradually increases the temperature, leading to bacterial cell membrane rupture and protein denaturation, thereby exerting its antibacterial properties. MXene, a two-dimensional nanosheet photothermal material, absorbs 808 nm near-infrared light and converts it into heat. Studies have demonstrated 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 osteoclast formation. Thus, MXene can both exert a highly effective antibacterial effect through PTT and inhibit osteoclast activity, making it a highly promising therapeutic agent for PAP.

[0024] For MXene to exert its bone resorption-inhibiting effect, it must reach the root apex. Previous studies have shown that, under the influence of an external magnetic field, the combination of Fe3O4 magnetic nanoparticles with traditional root canal sealers can increase the sealer's penetration into the dentinal tubules. Furthermore, Fe3O4 nanoparticles also inhibit bone resorption. Therefore, combining Fe3O4 nanoparticles with MXene holds great promise as a root canal disinfectant for PAP. After the MXene exerts its photothermal antibacterial effect within the root canal, the Fe3O4 nanoparticles, guided by the magnetic field, can deliver the MXene to the root apex to inhibit bone resorption. This provides a root canal disinfectant material that combines photothermal antibacterial and bone resorption inhibition. The Fe3O4-MXene root canal disinfectant utilizes the photothermal effect to inhibit bacteria, and the superparamagnetic nature of Fe3O4 allows for directional movement under the guidance of a magnetic field. Therefore, in addition to its effect within the root canal, the Fe3O4-MXene can also penetrate the root apex to inhibit alveolar bone resorption, thereby furthering the primary and ultimate goal of root canal treatment.

[0025] Example 1: Synthesis and characterization of Fe3O4-MXene Figure 1 As shown;

[0026] MXene Synthesis: MXene was synthesized using a previously reported method. The specific process was as follows: 1 g of LiF and 1 g of Ti3AlC2 MAX were dissolved in 20 mL of 9 M hydrochloric acid and magnetically stirred at 35°C for 24 hours. The reaction product was centrifuged at 3500 rpm and repeatedly washed with deionized water until the pH was ≥6. The MXene was then collected by centrifugation at the same speed and freeze-dried for later use.

[0027] Synthesis of Fe3O4 nanoparticles:

[0028] Fe₃O₄ nanoparticles were prepared using a solvothermal method. 0.69 g of FeCl₃·6H₂O, 0.24 g of trisodium citrate dihydrate, and 1.98 g of ammonium acetate were added sequentially to a polytetrafluoroethylene-lined reactor containing 36 mL of ethylene glycol. The mixture was stirred vigorously at room temperature for 1 hour. The reactor was then transferred to a 200°C oven and reacted for 16 hours. The product was washed with ethanol and water multiple times and then collected by magnetic separation.

[0029] Synthesis of Fe3O4-MXene:

[0030] 90 mg of MXene was dispersed in 60 mL of ethylene glycol and sonicated for 30 minutes. 0.27 g of FeCl₃·6H₂O, 0.972 g of 1,6-hexanediamine, and 1.08 g of sodium acetate were then added sequentially. After stirring for 1 hour, the mixture was transferred to a reactor and reacted at 200°C for 7 hours. The final product was magnetically separated and washed multiple times with ethanol and deionized water before being dispersed in deionized water.

[0031] Characterization of Fe3O4-MXene:

[0032] 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). Figure 2 As shown: (a) X-ray diffraction pattern (XRD): The characteristic peaks of MXene at 6.0° and 61.1° and the Fe3O4 standard diffraction peaks (JCPD 19-06290) of Fe3O4-MXene at 30.2° (200), 35.6° (311) and 56.2° (511) confirm the crystal structure integrity of the composite material. (b) Fourier transform infrared spectroscopy (FTIR): 560 cm −1 The enhanced Fe-O bond absorption peak at (e) confirms the successful Fe₃O₄ modification of MXene. (ce) Scanning electron microscopy and (fh) transmission electron microscopy: MXene exhibits a typical two-dimensional layered, wrinkled structure, while the Fe₃O₄ nanoparticles in the composite are uniformly distributed on the MXene surface. High-resolution TEM reveals clear Fe₃O₄ lattice fringes, and the MXene structure remains intact, confirming the successful synthesis of the composite. (i) Thermogravimetric analysis (TGA): The mass of Fe₃O₄ and Fe₃O₄-MXene decreases by 11.95% and 9.46%, respectively, demonstrating the excellent thermal stability of the composite. (j) Vibrating sample magnetometer (VSM): The magnetization of Fe₃O₄-MXene is 25 emu / g, and it moves under the attraction of a magnet within 10 seconds. (F: Fe₃O₄, M: MXene, FM: Fe₃O₄-MXene).

[0033] Example 2: Photothermal performance and biosafety evaluation of Fe3O4-MXene

[0034] Photothermal performance

[0035] First, 1 mL of Fe3O4-MXene solution was irradiated with near-infrared light (5.7 W / cm²), and the temperature change was recorded every minute using an infrared thermal imager. Subsequently, the absorbance of Fe3O4-MXene aqueous solutions with different concentrations in the near-infrared region was measured using a UV-visible spectrophotometer, and their photothermal properties were calculated and analyzed. In addition, five cycles of 10 minutes of illumination and 10 minutes of cooling were performed to evaluate the photothermal stability of Fe3O4-MXene under repeated illumination. Figure 3As shown: (ab) Temperature changes of Fe3O4-MXene with different concentrations after illumination: With the increase of Fe3O4-MXene concentration, 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-UV spectrum: With the increase of concentration, the absorption of near-infrared light by Fe3O4-MXene increased. (e) The extinction coefficient of Fe3O4-MXene at 808nm is 1.12 L / (g·cm). (f) Temperature-time curve of 320μg / mL Fe3O4-MXene after illumination, rising to the highest temperature and then naturally cooling. (g) According to Figure 3 .f calculated the time constant τ = 351.9s. Finally, Figure 3 The calculated photothermal conversion efficiency of Fe3O4-MXene is 45.67%. The above results show that Fe3O4-MXene has good photothermal performance.

[0036] Biosafety

[0037] Mouse fibroblast L929 cells with good growth status and normal morphology were selected, and after trypsin digestion, they were centrifuged at 1000 rpm for 1 min to collect the cells. The cells were seeded in a 96-well plate at a density of 5000 cells / well and cultured at 37°C and 5% CO2 for 24 hours to allow the cells to cover the bottom of the well plate. After removing the original culture medium, 100 μL of culture medium containing different concentrations of Fe3O4-MXene was added to each well of the experimental group, and 100 μL of normal culture medium was added to each well of the control group, and incubated at 37°C and 5% CO2 for 24 hours. Subsequently, the CCK-8 kit was used for cytotoxicity detection: the old culture medium was removed, the cells were rinsed three times with PBS, and 100 μL of culture medium and 10 μL of CCK-8 reagent were added to each well, and incubated in the dark at 37°C and 5% CO2 for 1 hour. The experimental setting contained only culture medium and CCK-8 solution as the blank group to avoid the influence of the color of the test solution and the culture medium itself. The supernatant was transferred to a new 96-well plate and the absorbance (OD) at a wavelength of 450 nm was measured using a multi-function enzyme-linked detector SpectraMax iD5.

[0038] Maxillary premolars with a single root canal without caries, root resorption or fracture were selected and preserved in 75% ethanol for 1 week. The crown was cut off at the enamel-cementum junction, retaining a root length of about 12 mm, and the working length was set at 0.5 mm at the root apex. All teeth were prepared to a #40 / 06 taper using ProTaper nickel-titanium rotary instruments and rinsed with 1.0% sodium hypochlorite. After root canal preparation, the sample was placed in a 1.5 mL sterile centrifuge tube containing 1 mL PBS and autoclaved at 121°C for 20 minutes. 0, 160 and 320 μg / mL of Fe3O4-MXene aqueous solution were injected into the root canal, respectively, and irradiated vertically with an 808 nm laser (5.7 W / cm²) for 10 minutes. The temperature changes at three locations outside the root canal (root canal orifice, middle root section and root apex) were recorded using a metal probe of a thermometer. If the temperature change of the outer surface of the root is less than 10°C, damage to the periodontal tissue can be avoided. Figure 4 As shown: (a) Compared with the control group, 20-640 μg / mL Fe₃O₄-MXene showed no significant cytotoxicity to L929 cells. (b) Fe₃O₄-MXene concentrations of 320 μg / mL and below did not cause root surface temperature changes exceeding 10°C after illumination, thus preventing thermal damage to periodontal tissues. Therefore, the maximum Fe₃O₄-MXene concentration used in subsequent experiments was 320 μg / mL.

[0039] Example 3: Photothermal antibacterial function testing of Fe3O4-MXene

[0040] Enterococcus faecalis culture

[0041] Prepare brain heart infusion broth (BHI) liquid medium: dissolve 14.8g of BHI powder in 400mL of deionized water, autoclave, cool, and store at room temperature until ready for use. Prepare BHI agar solid medium: dissolve 14.8g of BHI powder and 6g of agar powder in 400mL of BHI deionized water, autoclave, cool to 50°C, and pour onto plates. Prepare sugar-containing BHI medium: dissolve 14.8g of BHI powder and 4g of sucrose in 400mL of deionized water, autoclave, and sterilize thoroughly until ready for use. Enterococci were revived by streaking onto BHI agar plates and cultured overnight at 37°C in an anaerobic chamber (N2:90%, CO2:5%, H2:5%). Use an inoculating loop to pick a single colony and inoculate 10mL of BHI liquid medium. Culture overnight at 37°C in an anaerobic chamber (N2:90%, CO2:5%, H2:5%). Bacteria in the logarithmic growth phase were diluted to 1×10 8 CFU / mL is used for standby use. When culturing Enterococcus faecalis biofilm, dilute the bacterial solution to 2×10 6CFU / mL, and then transfer the bacterial solution to a 48-well plate, 500 μL per well, and culture for one week, changing the solution every other day, or add the bacterial solution to the prepared in vitro root canal, culture for 3 weeks, changing the solution every other day.

[0042] Inhibitory effect on planktonic Enterococcus faecalis

[0043] Diluted E. faecalis bacterial suspension was mixed with various concentrations of Fe₃O₄-MXene solutions and illuminated for 0, 5, and 10 minutes. After illumination, the E. faecalis bacteria were serially diluted, and 100 μL of the suspension was evenly spread onto a BHI solid plate. The plates were then incubated overnight at 37°C in an anaerobic chamber (N₂:90%, CO₂:5%, H₂:5%) and counted.

[0044] Inhibitory effect on Enterococcus faecalis biofilm

[0045] Remove the old culture medium and treat the E. faecalis biofilm according to the following groups: (1) PBS, (2) PBS+NIRirradiation, (3) Fe3O4-MXene, (4) Fe3O4-MXene+NIR irradiation, (5)1% NaClO, (6) Fe3O4-MXene+ NIR irradiation + 1% NaClO. The NaClO treatment method is: NaClO is allowed to act for 30 seconds, and then an equal volume of 5% mass fraction sodium thiosulfate is added to terminate the NaClO reaction. After the treatment, remove the supernatant, add BHI culture medium, scrape the E. faecalis at the bottom of the well plate for gradient dilution and plating. Alternatively, use 40#K file to collect the debris in the isolated root canal, transfer the debris to the ep tube containing BHI culture medium, and perform gradient dilution and plating. Figure 5 Figure 2: (a) Inhibitory effect of Fe3O4-MXene on planktonic Enterococcus faecalis. Significant inhibitory effect was achieved only after 10 minutes of exposure to 320 μg / mL Fe3O4-MXene. Therefore, subsequent experiments used 320 μg / mL for 10 minutes. (bc) Inhibitory effect of Fe3O4-MXene on Enterococcus faecalis biofilms in a well plate (b) and in an excised root canal (c): Fe3O4-MXene-mediated photothermal therapy effectively inhibited Enterococcus faecalis biofilms and further enhanced the antibacterial efficacy of NaClO, a traditional root canal disinfectant.

[0046] Example 4: Detection of the bone resorption inhibitory function of Fe3O4-MXene

[0047] RAW264.7 cells were induced to osteoclasts using α-MEM medium containing RANKL (100 ng / ml) in 48-well plates, with 1×10 cells seeded per well. 4 RAW264.7 cells were cultured and the medium (100 ng / ml RANKL) was changed on the 3rd day. Osteoclasts began to form on the 4th day.

[0048] Primary osteoclast induction: Bone marrow was collected from 4-week-old C57 mice, and 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. 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 cells per well. 4 Cells were cultured and the induction medium (50 ng / ml MCSF, 50 ng / ml RANKL) was replaced every other day; osteoclasts were obtained after 6-7 days.

[0049] Fe3O4-MXene treatment: RAW264.7 cells were co-cultured with different concentrations of Fe3O4-MXene and osteoclast induction was performed. Osteoclast differentiation was detected by tartrate-resistant acid phosphatase (TRAP) staining kit. Figure 6 Shown: (a) The main components of Fe3O4-MXene (C, O, Ti, and Fe) were detected on a glass slide of an isolated tooth root apex, demonstrating that Fe3O4-MXene can penetrate the apical foramen and reach the root apex under the influence of a magnetic field. (bc) The inhibitory effect of different concentrations of Fe3O4-MXene on osteoclasts derived from the RAW264.7 cell line. 40 μg / mL Fe3O4-MXene significantly inhibited osteoclast formation, indicating that only a small amount of material is required to reach the root apex to inhibit bone resorption. (c) The inhibitory effect of 40 μg / mL Fe3O4-MXene on primary osteoclasts.

[0050] The above embodiments merely illustrate the implementation methods of the present invention. Although the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that a person skilled in the art may make various modifications and improvements without departing from the scope of the present invention, and these modifications and improvements fall within the scope of protection of the present invention.

Claims

1. Application of Fe3O4-MXene material in the preparation of root canal disinfection material with photothermal antibacterial and bone resorption inhibition, characterized by: The specific steps of the preparation method of the Fe3O4-MXene material are as follows: Step 1: Synthesis of MXene: 1 g of LiF and 1 g of Ti3AlC2 MAX were dissolved in 20 mL of 9 M hydrochloric acid and magnetically stirred at 35°C for 24 h. The reaction product was centrifuged at 3500 rpm and repeatedly washed with deionized water until the pH was ≥ 6. Finally, the MXene was collected by centrifugation at the same speed and freeze-dried for later use. Step 2: Synthesis of Fe3O4-MXene: 90 mg of MXene was dispersed in 60 mL of ethylene glycol and ultrasonically treated for 30 minutes. 0.27 g of FeCl3·6H2O, 0.972 g of 1,6-hexanediamine, and 1.08 g of sodium acetate were added in sequence. After stirring for 1 hour, the mixture was transferred to a reactor and reacted at 200°C for 7 hours. The final product was magnetically separated and washed with ethanol / deionized water multiple times, and then dispersed in deionized water for use, thereby obtaining Fe3O4-MXene material.

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