Tetragonal-phase two-dimensional transition metal boride nanosheet as well as preparation method and application thereof

Abstract

The invention discloses a tetragonal-phase two-dimensional transition metal boride nanosheet and a preparation method and application thereof.The preparation method comprises the steps that a high-temperature solid-phase reaction is utilized, and a tetragonal-phase precursor is firstly prepared; adding the tetragonal phase precursor into a polytetrafluoroethylene reaction container containing 40% of hydrofluoric acid for liquid-phase topological chemical etching reaction, and magnetically stirring; after stirring, centrifuging to obtain a first precipitate, and freeze-drying the first precipitate to obtain tetragonal phase intermediate powder; adding the tetragonal-phase intermediate powder, hydrochloric acid and 40% hydrofluoric acid into a microwave hydrothermal reaction kettle, carrying out microwave-assisted hydrothermal etching reaction to peel an aluminum layer into a two-dimensional lamellar structure, rapidly heating to 120 DEG C, centrifuging after the reaction is finished to obtain a second precipitate, and intercalating the second precipitate by using a liquid-phase ultrasonic method to obtain a tetragonal-phase intermediate powder; the tetragonal phase two-dimensional transition metal boride is obtained. The tetragonal-phase two-dimensional transition metal boride nanosheet generates active oxygen under the action of ultrasound, and can be used for sonodynamic tumor treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of two-dimensional materials, and in particular to a tetragonal two-dimensional transition metal boride nanosheet, a preparation method and application thereof. Background Art

[0002] In recent years, with the development of science and technology, more and more cancer treatment methods have emerged and have been tried to be applied in actual tumor treatment, including photothermal therapy, photodynamic therapy, sonodynamic therapy, etc. Photothermal therapy is to kill tumor tissue by enriching photothermal agents in the tumor area and inducing thermal effects through lasers; the sonodynamic therapy process is to kill tumor tissue by converting oxygen molecules into singlet oxygen and other reactive oxygen species (ROS) in the tumor site under the induction of light by photosensitizers. However, due to the limited penetration depth of light into the human body, the therapeutic effect is often reduced due to insufficient penetration depth or attenuation of light intensity. Therefore, sonodynamic therapy using ultrasound can make up for the shortcomings of the light treatment process, and effectively kill tumor tissue by inducing sonosensitizers to produce reactive oxygen species with deep penetration depth. The sonosensitive therapeutic agent used in this treatment method has low toxicity in the absence of external ultrasound induction conditions, so it has low damage to normal tissues that have not been treated with ultrasound, ensuring accurate and effective tumor killing.

[0003] Currently, the sonosensitizers used are mostly organic molecules or organic-inorganic hybrid particles, which have good sonodynamic effects. However, these sonodynamic materials also have problems such as short retention time and poor stability, which weakens the actual tumor treatment effect or requires multiple doses to achieve effective treatment.

[0004] With the rapid development of the field of two-dimensional materials, various forms of two-dimensional materials have been developed. Many two-dimensional materials with excellent performance have provided new opportunities for improving the effect and stability of sonodynamic tumor treatment, and have great application potential in tumor treatment. Therefore, it is urgent to develop a two-dimensional material that has both good sonodynamic generation effect and stable properties, which is one of the key issues to further improve the effect of sonodynamic tumor treatment. Summary of the invention

[0005] The purpose of the present invention is to overcome the defect in the prior art that there is a lack of two-dimensional materials that have good sonodynamic generation effect, stable properties, and can be used to improve the sonodynamic tumor treatment effect.

[0006] In order to achieve the above object, the present invention provides a method for preparing tetragonal two-dimensional transition metal boride nanosheets, the preparation method comprising at least the following steps:

[0007] Step 1, uniformly mixing a transition metal, metallic aluminum, and elemental boron to obtain a mixture, placing the mixture in an argon atmosphere tube furnace for high-temperature solid-phase reaction to obtain a tetragonal phase precursor;

[0008] Step 2, adding the tetragonal phase precursor into a polytetrafluoroethylene reaction container containing 40% hydrofluoric acid to carry out a liquid phase topochemical etching reaction, and magnetically stirring for 4 hours; after the stirring is completed, centrifuging to obtain a first precipitate, and freeze-drying the first precipitate to obtain a tetragonal phase intermediate powder;

[0009] Step 3, adding the tetragonal phase intermediate powder, hydrochloric acid and 40% hydrofluoric acid into a microwave hydrothermal reactor for microwave-assisted hydrothermal etching reaction to peel off the aluminum layer into a two-dimensional lamellar structure, rapidly heating to 120°C and maintaining for 1 hour. After the reaction is completed, centrifuging to obtain a second precipitate, intercalating the second precipitate using liquid phase ultrasound to obtain a reaction product, and freeze-drying the reaction product to obtain a tetragonal two-dimensional transition metal boride.

[0010] Preferably, in step 1, the transition metal at least comprises molybdenum or tungsten.

[0011] Preferably, in step 1, the transition metal and elemental boron are in powder form, and the metallic aluminum is in powder form or flake form.

[0012] Preferably, in step 1, the ratio by weight of transition metal: metallic aluminum: elemental boron is (1:20:1) to (1:70:1).

[0013] Preferably, in step 1, the temperature of the high temperature solid phase reaction is 1400° C. to 1600° C., and the time is 10 h to 15 h.

[0014] Preferably, in step 2, the temperature of the liquid phase topochemical etching reaction is 20°C to 25°C, and the time is 6h to 12h; in step 3, the temperature of the microwave-assisted hydrothermal etching reaction is 90°C to 120°C, and the time is 1h to 3h.

[0015] The present invention also provides a tetragonal two-dimensional transition metal boride nanosheet prepared by the above preparation method.

[0016] Preferably, the size of the tetragonal two-dimensional transition metal boride nanosheet is 0.5 μm to 3 μm, and the thickness of the tetragonal two-dimensional transition metal boride nanosheet is 1 nm to 20 nm.

[0017] Preferably, the tetragonal two-dimensional transition metal boride nanosheets are MoAlB-MoB nanosheets or WAlB-WB nanosheets.

[0018] The present invention also provides an application of the above-mentioned tetragonal two-dimensional transition metal boride nanosheets, wherein the tetragonal two-dimensional transition metal boride nanosheets are used for preparing sonodynamic tumor therapeutic drugs.

[0019] Compared with the prior art, the beneficial effects of the technical solution of the present invention at least include:

[0020] (1) The present invention first prepares a tetragonal phase precursor through a high-temperature solid-phase reaction, then utilizes a liquid-phase topochemical etching reaction to obtain a tetragonal phase intermediate, and finally obtains a tetragonal phase two-dimensional transition metal boride through a microwave-assisted hydrothermal etching reaction. This method can retain the original crystal structure of the precursor and obtain a tetragonal phase two-dimensional transition metal boride nanosheet with a good lamellar structure. The good lamellar structure has the advantages of large specific surface area, obvious edge effect, good tropism arrangement, and easy processing. The tetragonal phase two-dimensional transition metal boride can generate active oxygen under the action of ultrasound, so it can be specifically treated for tumors through ultrasound induction.

[0021] (2) The constituent elements of the tetragonal two-dimensional transition metal boride of the present invention are all common trace elements in the human body, have good biocompatibility, and will not cause toxic side effects during the treatment process.

[0022] (3) The tetragonal two-dimensional transition metal boride of the present invention can be dispersed in any buffer solution or physiological saline suitable for clinical application, which is convenient for storage and application; the tetragonal two-dimensional transition metal boride can also be loaded in microneedles with hydrogen peroxide response function for local transdermal drug delivery. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The present invention is a flow chart for the preparation and synthesis of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets.

[0024] Figure 2 Characterization of the tetragonal precursor, tetragonal intermediate, and tetragonal two-dimensional transition metal boride of the present invention:

[0025] (A) Scanning electron microscope image of tetragonal phase precursor MoAlB; (B) Transmission electron microscope image of tetragonal phase precursor MoAlB; (C) Energy spectrum element distribution map of tetragonal phase precursor MoAlB; (D) Scanning electron microscope image of tetragonal phase intermediate Mo2AlB2; (E) Transmission electron microscope image of tetragonal phase intermediate Mo2AlB2; (F) Energy spectrum element distribution map of tetragonal phase intermediate Mo2AlB2; (G) Scanning electron microscope image of tetragonal phase two-dimensional transition metal boride MoAlB-MoB nanosheets; (H) Transmission electron microscope image of tetragonal phase two-dimensional transition metal boride MoAlB-MoB nanosheets; (I) Energy spectrum element distribution map of tetragonal phase two-dimensional transition metal boride MoAlB-MoB nanosheets.

[0026] Figure 3Characterization of the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder, dispersion, tetragonal precursor MoAlB, tetragonal intermediate Mo2AlB2, and tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet of the present invention:

[0027] (A) Tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder and dispersion; (B) X-ray diffraction (XRD) spectra of tetragonal precursor MoAlB, tetragonal intermediate Mo2AlB2, and tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder; (C) X-ray photoelectron spectroscopy (XPS) spectrum of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet.

[0028] Figure 4 The oxygen production performance and therapeutic effect on tumor cells of the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets of the present invention are shown in the figure:

[0029] (A) Reactive oxygen production curve of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets in an in vitro buffer system; (B) Reactive oxygen production rate diagram; (C) Green fluorescence staining diagram and intensity statistics of reactive oxygen produced by tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets in melanoma cells B16-F10; (D) Killing effect diagram of different concentrations of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets on melanoma cells B16-F10 under ultrasound / no ultrasound conditions; (E) Killing effect of different concentrations of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets on melanoma cells B19-F10 under ultrasound conditions, live / dead cell green / red fluorescence staining diagram.

[0030] Figure 5 The results of the co-culture of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets of different concentrations with normal cells L-929 are shown in the figure:

[0031] (A) Cell survival results when normal L-929 cells were co-cultured with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets of different concentrations; (B) Green / red fluorescence staining of live / dead cells when normal L-929 cells were co-cultured with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets of different concentrations.

[0032] Figure 6 The tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet microneedle of the present invention is:

[0033] (A) Microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets; (B) Hydrogen peroxide responsive release process of microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets; (C) Skin puncture ability of microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets.

[0034] Figure 7 This is a diagram showing the therapeutic effect of the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet of the present invention:

[0035] (A) Photo of the tumor of tumor-bearing mice after treatment with microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets and other treatment methods; (B) The weight of the tumor of tumor-bearing mice after treatment with microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets and other treatment methods; (C) HE staining section of the tumor of tumor-bearing mice after treatment with microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets and other treatment methods; (D) Tumor tissue sequencing results and q-PCR verification results of tumor-bearing mice after treatment with microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets and other treatment methods. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0037] Two-dimensional materials have special advantages due to their many unique properties: ① Adjustable electronic structure: Two-dimensional materials can control their electronic structure by adjusting the layer structure and composition, achieving controllable adjustment from conductor-semiconductor-insulator. This feature facilitates the regulation of the effect of sonodynamic reactive oxygen production; ② Large specific surface area and abundant reaction sites: Due to their size characteristics, two-dimensional materials have a large specific surface area and abundant reaction sites, providing sufficient reaction sites for various ROS production reactions or enzymatic reactions, which can effectively increase the probability of reaction occurrence and enhance the therapeutic effect; ③ Modifiable surface and stable properties: Many two-dimensional materials have abundant surface functional groups, which provide rich possibilities for surface modification or carrying molecules of such materials, making it easy to achieve organic-inorganic hybridization for functional design or stability improvement.

[0038] In recent years, the two-dimensional materials that have attracted much attention include graphene, transition metal sulfides (TMDs), transition metal carbon / nitrides (MXene), black phosphorus (BP), etc. Among them, the photothermal and photodynamic therapy functions of MXene have attracted the attention of many researchers. Because of its adjustable electronic structure and rich surface groups, it has shown good effects in the process of tumor treatment, making the potential of another MXene-like two-dimensional material transition metal boride (MBene) in the field of biological applications also attract the attention of researchers. Because its elemental composition contains transition metal elements and boron elements, its application in the biological field, including chemotherapy tumor treatment, radiotherapy tumor treatment, neutron capture therapy, immunotherapy, etc., may have better effects than MXene. However, there are few reports on the research of MBene in the field of biological applications. One of the main reasons is that its preparation process is relatively difficult, so there is no sufficient material basis to support its subsequent biological application research. Therefore, the synthesis of new two-dimensional transition metal boride MBene through advanced material preparation methods is of great scientific significance for expanding the application of two-dimensional materials in the biological field and exploring the therapeutic mechanism of two-dimensional materials.

[0039] In response to the above challenges, the present invention proposes a novel method for synthesizing two-dimensional transition metal boride and its application in sonodynamic therapy of tumors, wherein a transition metal boride precursor synthesized by a high-temperature solid-phase reaction is subjected to a two-step liquid-phase topochemical etching method to synthesize a two-dimensional transition metal boride MBene nanosheet: since the liquid-phase topochemical etching method adopted in this process does not destroy the crystal structure of the precursor, a thermodynamically metastable two-dimensional material can be prepared; since the prepared two-dimensional MBene nanosheet has a sonodynamic active oxygen generation effect and good biocompatibility, it is a good sonodynamic tumor treatment nanomedicine; since the obtained two-dimensional MBene nanosheet has the function of activating immunity, it can activate the immune function of the tumor area during the treatment process, thereby improving the tumor treatment effect.

[0040] The tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet is used as an example to explain in detail:

[0041] 1. Preparation of Tetragonal Two-Dimensional Transition Metal Boride MoAlB-MoB Nanosheets:

[0042] like Figure 1 As shown, the preparation method of the present invention consists of a two-step liquid phase topological chemical etching method, the precursor is MoAlB synthesized by high temperature solid phase reaction, the intermediate is Mo2AlB2, and the final product is MoAlB-MoB.

[0043] Step 1: Mix metal molybdenum powder, metal aluminum powder / sheet, and elemental boron powder in a ratio of 1:53.3:1 and add them to a high-temperature reaction boat. Place the high-temperature reaction boat in an atmosphere tube furnace, replace the atmosphere in the tube furnace with argon three times, and maintain the argon atmosphere for 30 minutes. Then, the tube furnace is heated to 1400°C at a heating rate of 150°C / h, kept at 1400°C for 12 hours, then cooled to 900°C at a rate of 60°C / h, and then cooled to room temperature at a rate of 180°C / h. At high temperatures, the metal aluminum powder / sheet melts, and the three solids can react inside the molten aluminum at high temperatures. After the reaction is completed, the reaction product is taken out, and the excess Al metal is dissolved with 3mol / L dilute hydrochloric acid. The remaining reaction product is centrifuged and washed with deionized water several times, and finally dried and ground to obtain the tetragonal phase precursor MoAlB powder.

[0044] Step 2: Add the tetragonal phase precursor MoAlB powder into a polytetrafluoroethylene reactor, and add 40% hydrofluoric acid for liquid phase topological chemical etching. The proportion of the tetragonal phase precursor MoAlB powder is 10wt%, the reaction temperature is room temperature, and the liquid phase etching reaction lasts for 3h to 12h. After the reaction is completed, the product is collected by centrifugation and washed with deionized water several times. After drying, the tetragonal phase intermediate Mo2AlB2 is obtained.

[0045] Step 3: Add the tetragonal intermediate Mo2AlB2 into a hydrothermal microwave reactor, and add a mixed acid prepared by 12mol / L hydrochloric acid and 40% hydrofluoric acid, wherein the hydrofluoric acid content is 30% / 50% / 70%, and the content of the tetragonal intermediate Mo2AlB2 is 2wt%. The reactor is heated to 120°C at 1200W in a microwave hydrothermal reactor, kept warm for 1h, and then cooled naturally. After the reaction is completed, the product is centrifuged and collected, and washed with deionized water for multiple times, and dried to obtain a tetragonal two-dimensional transition metal boride MoAlB-MoB powder.

[0046] Step 4: Re-disperse the above powder with water, perform liquid-phase ultrasonic intercalation for 2 hours using an ultrasonic probe, then collect the product by centrifugation, and obtain tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder after drying.

[0047] like Figure 2 As shown in (A), (B) and (C), the tetragonal phase precursor MoAlB obtained by high temperature solid phase reaction has a bulk structure with a size of about 5 μm, and the three elements Mo, Al and B are evenly distributed.

[0048] like Figure 2 As shown in (D), (E), and (F), the tetragonal phase intermediate Mo2AlB2 obtained by the first liquid phase topochemical etching has obvious traces of etching and obvious stratification, but the overall structure is still a block structure, and the three elements Mo, Al, and B are evenly distributed.

[0049] like Figure 2 As shown in (G), (H), and (I), the tetragonal phase intermediate Mo2AlB2 is subjected to microwave-assisted hydrothermal etching reaction and intercalation to obtain tetragonal phase two-dimensional transition metal boride MoAlB-MoB nanosheets, which have a good lamellar structure, are about 0.5μm to 3μm in size, and the three elements Mo, Al, and B are evenly distributed.

[0050] 2. Characterization of Tetragonal Two-Dimensional Transition Metal Boride MoAlB-MoB Nanosheets:

[0051] The structure and elements of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder were characterized by XRD and XPS.

[0052] like Figure 3 As shown in (A), the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets have a brown-black color and are easily dispersed in the aqueous phase.

[0053] like Figure 3 (B) shows that the precursor MoAlB obtained by high-temperature solid-phase reaction has good crystallinity, and its diffraction peak matches the standard card to a high degree, proving that the synthesis result of the tetragonal precursor MoAlB is good. After the first step of liquid phase etching, the crystal structure changes, and after the second liquid phase etching, obvious orientation arrangement characteristics appear, and the (020) diffraction peak moves toward the small angle direction, proving that the interplanar spacing increases, indicating that the two-step liquid phase etching successfully etches the tetragonal precursor MoAlB into tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets.

[0054] like Figure 3 From (C), we can see that the binding energy states of the three elements Mo, Al, and B in the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets are consistent with the bonding conditions of the tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets, and there are termination groups on the surface of the tetragonal MoAlB-MoB nanosheets.

[0055] 3. Characterization of the acoustic dynamic properties of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets:

[0056] Tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheet powder was dispersed in DMSO at a concentration of 500 mg / mL, and then MoAlB-MoB nanosheet dispersion was added to 2 mg / mL DPBF ethanol solution at a concentration of 200 μg / mL. The solution was ultrasonically treated with a probe (Suzhou Nason Ultrasonic Co., Ltd.) at an ultrasonic power of 3 W / cm 2The duty cycle was 50%, the ultrasonic time was 8 min, and the absorbance change of the solution was recorded every minute using an enzyme marker. The experimental process used hexagonal Mo 4 / 3B 2-x T z For comparison, the results are as follows Figure 4 As shown in (A), the rate of active oxygen generation is Figure 4 As shown in (B).

[0057] B16-F10 cells were cultured in a 96-well plate with DMEM + 10% FBS medium. When the cells reached a density of 50%, the medium was replaced with a medium containing 200 μg / mL MoAlB-MoB nanosheets. The other reference groups were blank control group, positive group, hexagonal Mo 4 / 3 B 2-x T z The drug-containing culture medium was added and incubated for 6 h before adding the reactive oxygen species staining probe. The ultrasound group was treated with ultrasound at a power of 3 W / cm 2 , the duty cycle was 50%, and the ultrasonic time was 3 minutes. After the ultrasonic treatment, the cells were incubated for 30 minutes and then observed using a fluorescent inverted microscope. The results are as follows Figure 4 As shown in (C).

[0058] B16-F10 cells were cultured in a 96-well plate with DMEM + 10% FBS medium. When the cells reached a density of 50%, the medium was replaced with a medium containing 200 μg / mL MoAlB-MoB nanosheets. The other reference groups were blank control group, hexagonal Mo 4 / 3 B 2-x T z The drug-containing culture medium was added and incubated for 6 h, and then the ultrasound group was treated with ultrasound at a power of 3 W / cm 2 The duty cycle was 50% and the ultrasonic time was 3 min. After the ultrasonic treatment, the cells were incubated for 30 min and then the cell counting kit (CCK-8) was used to count the number of cells. The results are shown in Figure 4 As shown in (D).

[0059] B16-F10 cells were cultured in a 96-well plate with DMEM + 10% FBS medium. When the cells reached a density of 50%, the medium was replaced with a medium containing 200 μg / mL MoAlB-MoB nanosheets. The other reference groups were blank control group, hexagonal Mo 4 / 3 B 2-x T z The drug-containing culture medium was added and incubated for 6 h, and then the ultrasound group was treated with ultrasound at a power of 3 W / cm 2, the duty cycle was 50%, and the ultrasonic time was 3 minutes. After the ultrasonic treatment, the cells were stained with a live / dead staining kit, where live cells were stained with Calcein-AM to show green fluorescence, and dead cells were stained with propidium iodide (PI) to show red fluorescence, so a fluorescent inverted microscope was used to observe the cell condition.

[0060] like Figure 4 As shown in (A) and (B), MoAlB-MoB nanosheets can generate active oxygen under ultrasound induction, and its generation rate is slightly higher than that of hexagonal Mo 4 / 3 B 2-x T z In the absence of ultrasonic stimulation, MoAlB-MoB nanosheets produce almost no reactive oxygen species, which can achieve specific tumor killing therapy.

[0061] like Figure 4 (C) shows that under ultrasonic induction, MoAlB-MoB nanosheets can produce reactive oxygen species, and the reactive oxygen species can enter the melanoma cells B16-F10 for the next step of cancer cell killing. In the absence of ultrasonic stimulation, MoAlB-MoB nanosheets hardly produce reactive oxygen species, which is consistent with the in vitro DPBF reactive oxygen species detection results.

[0062] like Figure 4 As shown in (D) and (E), the tetragonal MoAlB-MoB nanosheets can significantly kill melanoma cells B16-F10 under ultrasound, but have almost no killing effect on cells in the absence of ultrasound stimulation. 4 / 3 B 2-x T z , MoAlB-MoB nanosheets can achieve higher cancer cell clearance efficiency under the same conditions.

[0063] 4. Biosafety verification of tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets:

[0064] L-929 cells were cultured in 96-well plates with DMEM + 10% FBS medium. When the cells reached a density of 50%, the medium was replaced with a medium containing 200 μg / mL MoAlB-MoB nanosheets. The other reference groups were blank control group, hexagonal Mo 4 / 3 B 2-x T z Group, after adding the drug-containing culture medium and incubating for 24 hours, the number of cells was counted using a cell counting kit (CCK-8). The results are as follows Figure 5 As shown in (A).

[0065] L-929 cells were cultured in 96-well plates with DMEM + 10% FBS medium. When the cells reached a density of 50%, the medium was replaced with a medium containing 200 μg / mL MoAlB-MoB nanosheets. The other reference groups were blank control group, hexagonal Mo 4 / 3 B 2-x T z The cells were stained with a live / dead staining kit after the drug-containing culture medium was added and incubated for 24 hours. The live cells were stained with Calcein-AM to show green fluorescence, and the dead cells were stained with propidium iodide (PI) to show red fluorescence. Therefore, the cells were observed using a fluorescent inverted microscope. The results are as follows: Figure 5 As shown in (B).

[0066] like Figure 5 As shown in (A) and (B), MoAlB-MoB nanosheets did not show obvious toxicity to normal cells during co-culture with normal cells, proving that MoAlB-MoB nanosheet materials have good biosafety and no obvious toxic side effects.

[0067] 5. Preparation and characterization of microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets:

[0068] To achieve precise local transdermal drug delivery, MoAlB-MoB nanosheets were loaded into hydrogen peroxide-sensitive microneedles to achieve drug delivery. The microneedle matrix was made of PBA-HA material cross-linked with epigallocatechin-3-gallate (EGCG) that is responsive to hydrogen peroxide.

[0069] 200 mg of hyaluronic acid (HA) was added to a mixed solution of 4 mL of N, N-dimethylformamide and 6 mL of water, followed by the addition of 148 mg of 4-(4,6-dimethoxy[1.3.5]triazine-2-yl)-4-methylyl chloride (DMTMM) and 125 mg of 3-(aminomethyl)phenylborate hydrochloride (PBA). The solution was adjusted to pH 6.5 and stirred for 48 h. The solution was then dialyzed in deionized water for 3 days using a dialysis bag (MW: 3500) and then freeze-dried to obtain PBA-HA powder.

[0070] The PBA-HA powder obtained above was dissolved in deionized water at a concentration of 50 mg / mL, and then EGCG was added. The ratio of EGCG to PBA-HA was 1:1 / 1:2 / 1:3, respectively. After complete dissolution, MoAlB-MoB nanosheets were added for dispersion, and then dropped onto the microneedle template of polydimethylsiloxane (PDMS), centrifuged at 3500 rpm, and the microneedle tip was obtained. HA was used as the microneedle substrate. The results are shown in Figure 2. Figure 6 As shown in (A).

[0071] The hydrogen peroxide response release process of the microneedles was tested in PBS containing 5mmol / L H2O2. The fluorescent agent Rhodamine B was loaded on the microneedle tip, and the microneedles with different EGCG / PBA-HA ratios were immersed in PBS buffer and PBS containing 5mmol / LH2O2, respectively. The amount of rhodamine released by the microneedles was determined by the absorbance value of the solution, thereby measuring its hydrogen peroxide response release process. The results are shown in Figure 6 As shown in (B).

[0072] The puncture ability of microneedles was tested using commercially available pig skin as a substitute, and the puncture ability was evaluated by microneedle mechanical property test and puncture depth. First, the stress of the microneedle during the compression process was monitored using a material mechanical property tester to evaluate its puncture ability. Then, the fluorescent agent rhodamine B was loaded into the microneedle and punctured into pig skin. The maximum depth was observed using a laser confocal microscope. The results are as follows: Figure 6 As shown in (C).

[0073] like Figure 6 As shown in (A), the EGCG-PBA-HA microneedle tips loaded with MoAlB-MoB nanosheets have obvious drug aggregation phenomenon, and the overall structure of the microneedle is complete, the needle tips are neatly arranged, and effective drug loading can be achieved.

[0074] like Figure 6 As shown in (B), EGCG-PBA-HA microneedles loaded with MoAlB-MoB nanosheets can achieve responsive release to hydrogen peroxide, and the weaker the degree of cross-linking controlled by EGCG, the faster the release rate.

[0075] like Figure 6 As shown in (C), the EGCG-PBA-HA microneedles loaded with MoAlB-MoB nanosheets have good mechanical properties and can puncture pig skin, and the deepest puncture depth can reach 200 μm.

[0076] 6. Microneedles loaded with tetragonal two-dimensional transition metal boride MoAlB-MoB nanosheets were used for in vivo treatment of melanoma-bearing mice:

[0077] B16-F10 melanoma cells were cultured with DMEM + 10% FBS medium, and each mouse was injected with 10 6 B16-F10 melanoma cells were used to establish a subcutaneous tumor-bearing model in C57BL / 6 mice. 3 The tumor-bearing mice were randomly divided into eight groups:

[0078] (1) Modeling group;

[0079] (2) blank microneedle group, microneedles without drug loading;

[0080] (3) ultrasound group, only ultrasound treatment;

[0081] (4) Blank microneedle + ultrasound, using microneedles without drug loading for ultrasound treatment;

[0082] (5) Six-party conflict 4 / 3 B 2-x T z Microneedle group, using microneedles to deliver hexagonal phase Mo 4 / 3 B 2-x T z Get treatment;

[0083] (6) Six-party conflict 4 / 3 B 2-x T z Microneedle + ultrasound group, using microneedles to deliver hexagonal phase Mo 4 / 3 B 2-x T z Perform sonodynamic therapy;

[0084] (7) Tetragonal MoAlB-MoB nanosheet microneedle group, using microneedles to deliver tetragonal MoAlB-MoB nanosheets for treatment;

[0085] (8) Tetragonal MoAlB-MoB nanosheet microneedle + ultrasound group, using microneedles to deliver tetragonal MoAlB-MoB nanosheets for sonodynamic therapy;

[0086] Tetragonal MoAlB-MoB nanosheets and hexagonal Mo 4 / 3 B 2-x T z The drug dispersion was locally administered by microneedle loading. The drug dosage was determined according to the weight of the mouse, which was 5 mg / kg. Ultrasound induction was performed 2 hours after the microneedle was inserted into the tumor area. The ultrasound power was 3 W / cm 2 The duty cycle was 50%, and the ultrasound time was 3 minutes. Two treatments were performed on the seventh and ninth days after tumor inoculation. The tumor tissues of the mice were then sampled, photographed and weighed. The results are as follows: Figure 7 (A) and (B) show that the tumor tissue of mice was taken for HE staining and sectioning to observe the inhibition of tumor cells. The results are shown in Figure 7 As shown in (C); RNA sequencing was performed on mouse tumor tissues, and the sequencing results were verified by qPCR. The results are shown in Figure 7 As shown in (D).

[0087] like Figure 7 As shown in (A), the tetragonal MoAlB-MoB nanosheets delivered by microneedles can effectively inhibit the growth of melanoma after ultrasound induction.

[0088] like Figure 7 As shown in (B), after all mice were killed, the ex vivo tumor tissues were taken out and weighed. Among them, the tumor weight of the tetragonal MoAlB-MoB nanosheets delivered by microneedles was the smallest after ultrasound induction, indicating that it can effectively inhibit tumor growth.

[0089] like Figure 7 As shown in (C), the tetragonal MoAlB-MoB nanosheets delivered by microneedles can regulate the reactive oxygen-related pathways to kill tumors after ultrasound-induced therapy, and can induce immune system response, thereby enhancing its therapeutic effect on tumors.

[0090] like Figure 7 As shown in (D), the sequencing result pathway diagram shows that the treatment process activated the reactive oxygen species-related pathway, which is consistent with the mechanism of the treatment process.

[0091] In summary, the present invention provides a method for preparing a tetragonal two-dimensional transition metal boride nanosheet, comprising: synthesizing a tetragonal phase precursor by a high-temperature solid-phase reaction, obtaining a tetragonal phase intermediate by a liquid-phase topological chemical etching reaction, and then obtaining a tetragonal phase two-dimensional transition metal boride nanosheet retaining the original crystal structure by a microwave-assisted hydrothermal etching reaction; the tetragonal phase two-dimensional transition metal boride nanosheet can produce reactive oxygen species under ultrasonic stimulation, and can effectively kill tumor cells; its constituent elements are all trace elements of the human body, have good biocompatibility, and can be used as a sonodynamic therapeutic drug to treat tumors, and its good sonodynamic reactive oxygen generation efficiency makes it have a stronger tumor inhibition effect than the existing hexagonal structured nanosheets.

[0092] 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 tetragonal two-dimensional transition metal boride nanosheets, characterized in that: The preparation method at least comprises the following steps: Step 1, uniformly mixing a transition metal, metallic aluminum, and elemental boron to obtain a mixture, placing the mixture in an argon atmosphere tube furnace for high-temperature solid-phase reaction to obtain a tetragonal phase precursor; Step 2, adding the tetragonal phase precursor into a polytetrafluoroethylene reaction container containing 40% hydrofluoric acid to carry out a liquid phase topochemical etching reaction, and magnetically stirring for 4 hours; after the stirring is completed, centrifuging to obtain a first precipitate, and freeze-drying the first precipitate to obtain a tetragonal phase intermediate powder; Step 3, adding the tetragonal phase intermediate powder, hydrochloric acid and 40% hydrofluoric acid into a microwave hydrothermal reactor for microwave-assisted hydrothermal etching reaction to peel off the aluminum layer into a two-dimensional lamellar structure, rapidly heating to 120°C and maintaining for 1 hour. After the reaction is completed, centrifuging to obtain a second precipitate, intercalating the second precipitate using liquid phase ultrasound to obtain a reaction product, and freeze-drying the reaction product to obtain a tetragonal two-dimensional transition metal boride.

2. The preparation method according to claim 1, characterized in that In step 1, the transition metal at least includes molybdenum or tungsten.

3. The preparation method according to claim 1, characterized in that: In step 1, the transition metal and elemental boron are in powder form, and the metallic aluminum is in powder form or flake form.

4. The preparation method according to claim 1, characterized in that: In step 1, the ratio by weight of transition metal: metallic aluminum: elemental boron is (1:20:1) to (1:70:1).

5. The preparation method according to claim 1, characterized in that: In step 1, the temperature of the high temperature solid phase reaction is 1400° C. to 1600° C., and the time is 10 h to 15 h.

6. The preparation method according to claim 1, characterized in that: In step 2, the temperature of the liquid phase topological chemical etching reaction is 20° C. to 25° C., and the time is 6 h to 12 h; in step 3, the temperature of the microwave-assisted hydrothermal etching reaction is 90° C. to 120° C., and the time is 1 h to 3 h.

7. A tetragonal two-dimensional transition metal boride nanosheet, characterized in that: The tetragonal two-dimensional transition metal boride nanosheet is prepared by the preparation method described in any one of claims 1 to 6.

8. The tetragonal two-dimensional transition metal boride nanosheet according to claim 7, characterized in that: The size of the tetragonal two-dimensional transition metal boride nanosheet is 0.5 μm to 3 μm, and the thickness of the tetragonal two-dimensional transition metal boride nanosheet is 1 nm to 20 nm.

9. The tetragonal two-dimensional transition metal boride nanosheet according to claim 7, characterized in that: The tetragonal two-dimensional transition metal boride nanosheets are MoAlB-MoB nanosheets or WAlB-WB nanosheets.

10. A use of the tetragonal two-dimensional transition metal boride nanosheet according to any one of claims 7 to 9, characterized in that: The tetragonal two-dimensional transition metal boride nanosheet is used for preparing sonodynamic tumor therapeutic drugs.

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