A two-dimensional bionic titanium diboride nanomaterial and its preparation method and application

Abstract

The present invention discloses a two-dimensional bionic titanium diboride nanomaterial for photothermal and / or sonodynamic treatment of tumors, characterized in that the two-dimensional bionic titanium diboride nanomaterial is obtained by coating titanium diboride nanosheets with cell membranes and modifying phospholipid-polyethylene glycol-sequence peptides; the size of the titanium diboride nanosheets is 50-100nm, and the cell membrane has the characteristic of being able to pass through the blood-brain barrier. The present invention prepares a two-dimensional bionic nanomaterial with better photothermal and sonodynamic synergy for penetrating the blood-brain barrier by coating the surface of the two-dimensional TiB2 nanosheets with cell membranes, and further modifying phospholipid-polyethylene glycol-RGD to anchor the targeting peptide, and enhances the enrichment and targeting ability of the material at the tumor site, and further enhances the photothermal and sonodynamic synergistic treatment effect of the tumor, effectively realizing the precise treatment of brain gliomas.

Description

Technical Field

[0001] The invention belongs to the field of nano material photothermal reagents or sonosensitizers, and particularly relates to a two-dimensional bionic titanium diboride nano material and a preparation method and application thereof. Background Art

[0002] Glioma (GBM) is one of the most aggressive and lethal tumors with the shortest median survival time and the lowest five-year survival rate. On the one hand, from the perspective of treatment, surgical resection of primary tumor tissue followed by adjuvant radiotherapy or chemotherapy is the main treatment strategy for GBM. However, the high invasiveness of GBM to normal tissue limits the precise resection of tumor tissue. There is an urgent need to develop effective treatments for GBM. Sonodynamic therapy (SDT) is a new cancer treatment strategy that uses ultrasound (US) to stimulate sonosensitizers to produce a large amount of reactive oxygen species (ROS) to kill malignant tumors.

[0003] Sonodynamic therapy (SDT) uses harmless ultrasound (US) as an energy source, which can effectively excite sonosensitizers accumulated in situ in tumors to produce highly toxic reactive oxygen species (ROS) to destroy tumor cells without causing any observable side effects to normal tissues. Sonosensitizers play a vital role in the SDT process. Traditional organic sonosensitizers derived from photosensitizers (such as phthalocyanine, hematoporphyrin, and porphyrin derivatives) are usually phototoxic to the skin and face disadvantages such as poor chemical stability, low bioavailability, and low tumor accumulation. The most representative example of inorganic sonosensitizers is titanium dioxide (TiO2)-based nanomaterials, but their ROS quantum yield activated by US is relatively low due to the rapid integration of electrons and holes, resulting in poor sonodynamic therapeutic effects of such traditional sonosensitizers.

[0004] On the other hand, the prognosis of GBM is usually poor, mainly due to tumor heterogeneity, drug resistance, insufficient drug delivery to brain tumors, etc. The blood-brain barrier (BBB) ​​and blood-brain tumor barrier (BBTB) prevent more than 98% of therapeutic agents from entering the brain tumor site, but the difficulties in overcoming the BBB and identifying target cells greatly limit the therapeutic efficiency of GBM. Therefore, the preparation of multifunctional nanomedicines with efficient BBB penetration and enhanced GBM targeting ability is very important for the effective treatment of GBM. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a two-dimensional bionic titanium diboride nanomaterial and its preparation method and application in view of the deficiencies of the above-mentioned prior art. The present invention realizes effective tumor targeting by coating the surface of two-dimensional TiB2 nanosheets with cell membranes and modifying them with phospholipid-polyethylene glycol-sequence peptides, overcomes the limitation of nanomaterials in crossing the BBB, and further enhances the synergistic effect of tumor photothermal and sonokinetic therapy.

[0006] The technical solution adopted by the present invention to solve the above-mentioned problems is:

[0007] A two-dimensional bionic titanium diboride nanomaterial is obtained by coating titanium diboride nanosheets (TiB2 NSs) with cell membranes and modifying them with phospholipid-polyethylene glycol-sequence peptides; the size of the titanium diboride nanosheets is 50-100nm.

[0008] According to the above scheme, the thickness of the titanium diboride nanosheet is 0.5-2 nm, and the thickness of the cell membrane is generally 2-10 nm.

[0009] According to the above scheme, the titanium diboride nanosheets are prepared by a liquid phase exfoliation method, and a special defect structure is formed during the preparation process and the surface is partially oxidized to TiO2, which can be used as an efficient sonosensitizer to generate reactive oxygen species (ROS) under ultrasonic irradiation. Further, the preparation method of the titanium diboride nanosheets includes the following steps:

[0010] S1. Evenly dispersing titanium diboride powder in water to obtain a titanium diboride aqueous solution with a concentration of 0.1-1 mg / mL; the titanium diboride powder is micron-sized titanium diboride block particles;

[0011] S2, placing the titanium diboride aqueous solution obtained in S1 into a microwave synthesizer for microwave synthesis, and the supernatant obtained by centrifugation after the reaction is a titanium diboride nanosheet (TiB2 NSs) dispersion. After the titanium diboride nanosheet dispersion is dried, titanium diboride nanosheets can be obtained, which can be dispersed in water to prepare an aqueous solution of corresponding concentration when used. Among them, the microwave synthesis temperature is 80-110°C, the power is 10-50w, and the time is 10-30min; the centrifugal speed is 8500-10000 rpm, and the time is 10-30min.

[0012] The method for preparing the above two-dimensional bionic titanium diboride nanomaterial comprises the following steps:

[0013] (1) Preparation of titanium diboride nanosheets (TiB2 NSs) dispersion by liquid phase exfoliation method;

[0014] (2) mixing the titanium diboride nanosheets (TiB2 NSs) dispersion with the cell membrane and performing ultrasound, centrifuging the mixture after ultrasound to remove excess cell membrane in the supernatant, and redispersing the resulting precipitate in deionized water to obtain a cell membrane-coated titanium diboride nanosheet (TiB2@CM) dispersion;

[0015] (3) The cell membrane-coated titanium diboride nanosheet (TiB2@CM) dispersion is mixed with phospholipid-polyethylene glycol-sequence peptide (phospholipid-polyethylene glycol-RGD) in a certain mass ratio, and reacted in a constant temperature oscillator for a period of time to obtain a two-dimensional bionic titanium diboride nanomaterial (TiB2@CM-RGD).

[0016] According to the above scheme, in step (1), the concentration of the TiB2 NSs dispersion is 0.05-0.15 mg / mL, the size of the TiB2 NSs is 50-100 nm, the thickness is 0.5-2 nm, it has good near-infrared II (NIRⅡ) absorption, and has good sonodynamic properties as a titanium-based nanomaterial.

[0017] According to the above scheme, in step (2), the mass ratio of TiB2 NSs to cell membrane is 4:1-2:1, and the ultrasonic time is 30-60 min.

[0018] According to the above scheme, in step (3), the mass ratio of TiB2@CM to phospholipid-polyethylene glycol-RGD is 1:1-3:1; the temperature of the constant temperature box is 37-55°C, and the shaking speed is 100-150 rpm.

[0019] According to the above scheme, the cell membrane is extracted by using a kit after cell lysis, and has the characteristic of being able to pass through the blood-brain barrier. The cell can be one or more of mouse breast cancer cells (4T1 cells), human glioma cells (U87 cells), macrophages (J774A.1 cells), etc., preferably J774A.1.

[0020] According to the above scheme, the phospholipid-polyethylene glycol-RGD can specifically be DSPE-PEG2000-RGD or the like.

[0021] The two-dimensional biomimetic titanium diboride nanomaterials of the present invention can be used as agents for photothermal and / or sonodynamic therapy of tumors, especially gliomas of the brain glial column. Among them, sonodynamic therapy (SDT) uses ultrasound, and the specific conditions are 1-2.5w / cm 2 , ultrasound time is 1-10min; the laser conditions for photothermal therapy (SDT) are: 808-1064nm, direct irradiation time is 1-10min; the dispersion concentration of two-dimensional bionic titanium diboride nanomaterials as tumor treatment agents in cells, tissues or living bodies is 5-15mg / mL.

[0022] The technical concept of the present invention is as follows:

[0023] Firstly, the present invention adopts a microwave synthesis method to obtain a two-dimensional titanium diboride nanosheet as a raw material, which forms a special defect structure and partially oxidizes its surface to TiO2 during the liquid phase exfoliation process. It can also be used as a highly efficient sonosensitizer to produce reactive oxygen species (ROS) under ultrasonic irradiation for ultrasonic dynamic treatment of cancer. At the same time, the titanium diboride nanosheet has a high photothermal conversion efficiency in the near-infrared II region. Under NIR II laser irradiation, the TiB2 nanosheet produces mild photothermal heating to the tumor, promotes blood flow in the tumor, improves tumor oxygenation, and can achieve the synergistic effect of near-infrared II region photothermal therapy and sonodynamic therapy, thereby achieving the purpose of tumor ablation in deeper tissues.

[0024] Second, in order to overcome the limitation of nanomaterials in crossing the BBB, the present invention uses a bionic cell membrane (CM) to coat the surface of the two-dimensional TiB2 nanosheet and modifies the phospholipid-polyethylene glycol-RGD anchor peptide segment on its surface to enhance the enrichment of nanomaterials in brain glioma. The two-dimensional TiB2 nanosheet coated with the cell membrane can well maintain the targeting effect of cells that depend on specific proteins on the membrane, thereby achieving effective tumor targeting. Taking the macrophage (J774.A1) membrane selected by the present invention as an example, on the one hand, the macrophage membrane can effectively disguise the nanomaterial so that it is not cleared by the immune system; on the other hand, the α4, β1 protein and macrophage 1 antigen (Mac-1) overexpressed on the surface of macrophages enable them to penetrate the BBB to effectively target glioma. Then, the present invention further modifies DSPE-PEG2000-RGD on the surface of the cell membrane, and the surface anchoring of the peptide segment further enhances the targeting effect of tumor cells, significantly enhances the brain enrichment effect of nanomaterials, thereby achieving a stronger BBB penetration ability and further eliminating brain glioma.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention prepares a two-dimensional bionic nanomaterial with photothermal and sonicotinic synergy that has better blood-brain barrier penetration effect by coating the surface of the two-dimensional TiB2 nanosheet with cell membrane and further modifying phospholipid-polyethylene glycol-RGD to anchor the targeting peptide, thereby enhancing the enrichment and targeting ability of the material at the tumor site, and further enhancing the photothermal and sonicotinic synergistic treatment effect of the tumor, effectively realizing the precise treatment of brain glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a scanning electron microscope image of titanium diboride powder before peeling;

[0028] Figure 2 This is a transmission electron microscope image of a two-dimensional TiB2 nanosheet;

[0029] Figure 3 This is an atomic force microscope image of a two-dimensional TiB2 nanosheet;

[0030] Figure 4 is the XRD diffraction pattern of two-dimensional TiB2 nanosheets;

[0031] Figure 5 This is the XPS result of two-dimensional TiB2 nanosheets;

[0032] Figure 6 Characterization of TiB2 NSs, TiB2@CM and TiB2@CM-RGD, where Figure 6 a is the transmission electron microscope image of TiB2@CM, Figure 6 b is the zeta potential change of TiB2 NSs, TiB2@CM and TiB2@CM-RGD;

[0033] Figure 7 The study shows the photothermal conversion efficiency of two-dimensional TiB2 nanosheets. a is the heating curve of two-dimensional TiB2 nanosheets with different concentrations; b is the heating curve of 0.3mg / mL two-dimensional nanomaterial TiB2 under different powers of 1064nm laser. c is the heating curve of two-dimensional nanomaterial TiB2 (0.6mg / mL, 1W / cm 2 , 1064nm) after 5 cycles of heating and cooling; d is the Lambert-Beer absorption coefficient at a wavelength of 1064nm; e is the cooling curve and photothermal conversion efficiency of the two-dimensional nanomaterial TiB2 at 1064nm.

[0034] Figure 8 Research showing the photothermal conversion efficiency of two-dimensional nanomaterial TiB2. Among them, a is the heating curve of two-dimensional nanomaterial TiB2 with different concentrations; b is the heating curve of 0.3mg / mL two-dimensional nanomaterial TiB2 under 808nm laser with different powers; c is the Lambert Beer absorption coefficient at a wavelength of 808nm; d is the cooling curve and photothermal conversion efficiency of two-dimensional nanomaterial TiB2 at 808nm.

[0035] Fig. 9 The temperature change evaluation of two-dimensional nanomaterial TiB2 at different penetration depths under the action of 808nm and 1064nm lasers is shown. Among them, a is a schematic diagram of the device; b and c are photothermal imaging and temperature changes in the aqueous suspension of two-dimensional nanomaterial TiB2 after 808nm laser and 1064nm laser penetrated different tissue depths.

[0036] Fig.10 The acoustic dynamic performance of two-dimensional nanomaterial TiB2 was investigated. Among them, a and b are the acoustic dynamic performance of commercial TiO2 and two-dimensional nanomaterial TiB2 under ultrasound (40kHz, 3.0W / cm 2,50%duty cycle)DPBF degradation over time; c is the degradation rate constant of DPBF under ultrasound for commercial TiO2 and two-dimensional nanomaterial TiB2; d is the comparison of the sonodynamic properties of two-dimensional nanomaterial TiB2 and commercial TiO2.

[0037] Fig.11 The biosafety and cell therapy effect of the two-dimensional bionic nanomaterial TiB2@CM-RGD are shown. Among them, a is the cell activity of different cells (U87 human brain astroglioma cells, MCF-7 human breast cancer cells, HaCat human immortalized epidermal cells) after the two-dimensional bionic nanomaterial TiB2@CM-RGD was co-incubated with different cells for 4 hours; b is the cell activity after different treatments (a: PBS group; b: PBS+ultrasound group; c: PBS+photothermal group; d: PBS+ultrasound+photothermal group; e: TiB2@CM-RGD group; f: TiB2@CM-RGD+photothermal group; g: TiB2@CM-RGD+ultrasound group; h: TiB2@CM-RGD+ultrasound+photothermal group); c is the production of reactive oxygen species in different groups after different treatments, as well as the staining results of live cells (green fluorescence) and dead or late apoptotic cells (red fluorescence).

[0038] Fig.12 The possibility of the two-dimensional bionic nanomaterial TiB2@CM-RGD being enriched in the brain. Among them, a is the fluorescence imaging of two-dimensional bionic nanomaterial TiB2@CM-RGD at different concentrations; b is the correlation between two-dimensional bionic TiB2@CM-RGD nanomaterials at different concentrations and fluorescence imaging intensity; c is the changes in fluorescence imaging of brain tumor sites within 48 hours after in vivo injection of different materials (TiB2, TiB2@CM, TiB2@CM-RGD).

[0039] The above drawings are all corresponding drawings involved in the embodiments. DETAILED DESCRIPTION

[0040] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

[0041] In the present invention, the titanium diboride powder used is a micron-sized titanium diboride powder, and its microscopic morphology is block-shaped particles. The titanium diboride powder specifically used in the following embodiment 1 is as follows: Figure 1 Shown are bulk particles of titanium diboride with a size of 1-20 μm.

[0042] Example

[0043] 1. Preparation of two-dimensional bionic nanomaterial TiB2@CM-RGD.

[0044] (1) Disperse 5 mg of titanium diboride powder in water and treat with ultrasound for 1 h to uniformly disperse titanium diboride in the aqueous solution to obtain a 1 mg / mL titanium diboride aqueous solution. Then, place the aqueous solution in a microwave synthesizer for reaction for 15 min (110°C, 50 W / cm -2 ), then turn off the microwave synthesizer, allow the solution to cool naturally to room temperature, take out the product, and centrifuge (8000 rpm / min, 10 min) to obtain the supernatant, which is the titanium diboride nanosheets (TiB2 NSs) dispersion (concentration of 0.1 mg / mL).

[0045] (2) J774A.1 cells were lysed and their cell membranes were extracted using a kit to obtain a cell extract (concentration of 0.05 mg / mL) which was stored at -80°C.

[0046] According to the mass ratio of TiB2 NSs to cell membrane of 4:1, the cell membrane extract and TiB2 NSs dispersion were mixed and ultrasonicated for 30 min. The mixture after ultrasonication was centrifuged to remove the excess cell membrane in the supernatant, and the precipitate was redispersed in deionized water to obtain the TiB2@CM dispersion (concentration of 0.1 mg / mL).

[0047] According to the mass ratio of TiB2@CM to DSPE-PEG2000-RGD being 2:1, the above TiB2@CM dispersion was mixed with DSPE-PEG2000-RGD and reacted in a constant temperature oscillator for 1 hour (37°C, 150rpm / min) to obtain the two-dimensional bionic titanium diboride nanomaterial TiB2@CM-RGD.

[0048] 2. Characterization of two-dimensional biomimetic titanium diboride nanomaterials TiB2@CM-RGD

[0049] like Figure 2 Transmission electron microscope (TEM) images show that the TiB2 prepared by the liquid phase exfoliation method is a lamellar structure with a size of 50-100nm; Figure 3 Atomic force microscopy (AFM) showed that the thickness of the TiB2 nanosheets was about 1.2 nm. XRD analysis was performed to determine the composition, purity and crystal structure of the obtained TiB2 nanosheets. Figure 4 As shown in Figure 3, the characteristic diffraction peaks with 2θ values ​​of 27.587°, 34.132°, 44.437°, 56.991°, 61.104°, 68.326° and 78.639° correspond to (001), (100), (101), (002), (110), (111) and (201) respectively (PDF#35-0741), and almost no diffraction peaks of other impurities were observed, indicating that the purity of the obtained TiB2 nanosheets is very high. Figure 5X-ray photoelectron spectroscopy (XPS) shows that the measured spectrum of TiB2 nanosheets shows the presence of typical Ti and B signals. The above shows that TiB2 nanosheets were successfully prepared via liquid phase exfoliation.

[0050] TEM image of TiB2@CM-RGD negatively stained with uranyl acetate Figure 6 As shown in a, the TiB2 nanosheets are uniformly covered by a layer of cell membrane, which is about 50nm in size and about 4nm in thickness. Figure 6 As shown in b, after the TiB2 nanosheets were surface-modified with cell membrane and DSPE-PEG2000-RGD, the surface potential gradually became negative, indicating the successful preparation of the two-dimensional bionic titanium diboride nanomaterial TiB2@CM-RGD.

[0051] 3. Study on the photothermal properties of two-dimensional nanomaterial TiB2

[0052] The photothermal performance of 2D TiB2 nanosheets with different concentrations was evaluated under 808nm and 1064nm laser irradiation with different power densities. Figure 7 a, b and 8a, b: The photothermal effect of two-dimensional TiB2 nanosheets depends on the concentration of the material and the power density of the light. Typically, under a 1064 nm laser, 0.6 mg / mL of TiB2 NSs is heated by 1 W / cm 2 After laser irradiation, the temperature can rise by 38°C. Under 808nm laser, 0.6mg / mL TiB2 NSs was irradiated with 1W / cm 2 After laser irradiation, the temperature can rise by 42°C. Figure 7 d, 8c The extinction coefficient of the two-dimensional nanomaterial TiB2 NSs was determined. According to the Lambert-Beer law (A / L = εC, where ε is the extinction coefficient), the linear dependence of A / L on concentration was obtained, and the extinction coefficients at 808 and 1064 nm were measured to be 3.26 and 3.27 L g, respectively. -1 cm -1 In addition, the photothermal conversion efficiency (η) of the two-dimensional nanomaterial TiB2 NSs was calculated based on the results of the heat transfer time constant and the maximum steady-state temperature ( Figure 7 e, 8d), the value is as high as 32.9% at 808nm and 37.6% at 1064nm. Figure 7 c, After 5 laser on-off cycles, TiB2 NSs exhibited high photothermal stability, ensuring sustainable / repeatable photothermal therapy.

[0053] like Fig. 9 As shown in Figure 2, the temperature changes of TiB2 NSs were detected after tissue penetration of chicken breasts of different thicknesses (0, 2, 4, 6, 8, and 10 mm) under 808 and 1064 nm laser irradiation. Fig. 9 a is a schematic diagram of the device. 300 μL of dispersed TiB2 NSs aqueous suspension (0.3 mg / mL) was covered with chicken breast tissue of different thicknesses and irradiated with 808 and 1064 nm lasers (1 W / cm 2 ) for 5 minutes, and then use a thermal imager to take infrared thermal images ( Fig. 9 b) and record the temperature change of TiB2 NSs water suspension under chicken breast of different thicknesses ( Fig. 9 c) It can be observed that under 808nm and 1064nm laser radiation, when penetrating tissue of the same thickness, the use of 1064nm laser can achieve a better heating effect than the use of 808nm laser.

[0054] 4. Study on the acoustic dynamic properties of two-dimensional nanomaterial TiB2

[0055] In order to evaluate the sonodynamic properties of TiB2 NSs, the commonly used 1,3-diphenylisobenzofuran (DPBF) molecular probe was used to monitor the reactive oxygen species (ROS) generated by TiB2 NSs triggered by ultrasound. The generated ROS can react with DPBF, resulting in a decrease in the characteristic absorption at 416nm in the UV-visible-near infrared spectrum. The specific operation was to mix 100μL (0.1mg / mL) TiB2 NSs aqueous solution, commercial TiO2 NPs aqueous solution and 2mL (50μg / mL) DPBF ethanol solution, and then perform ultrasonic treatment. The ultrasonic conditions were: 40kHz, 3.0W / cm 2 , 50% duty cycle.

[0056] like Fig.10 As shown in a and b, in the presence of TiB2 NSs, the characteristic peak intensity of DPBF at 416 nm was significantly reduced, indicating that TiB2 NSs can produce a large amount of ROS under ultrasonic irradiation. Compared with commercial TiO2 nanoparticles (TiO2 NPs), TiB2 NSs were found to have stronger DPBF oxidation ability under the same ultrasonic conditions, indicating that TiB2 NSs have stronger sonosensitization efficiency than commercial TiO2NPs ( Fig.10 d) In addition, the rate constant of DPBF oxidation in the presence of TiB2 NSs under ultrasound was investigated. Fig.10 c shows that the rate constant is 0.0360min -1 , which is significantly higher than the 0.0159 min corresponding to TiO2 NPs. -1 .

[0057] 5. Study on the biosafety and tumor cell killing effect of two-dimensional bionic nanomaterial TiB2@CM-RGD

[0058] Based on the strong photothermal conversion ability and efficient sonodynamic performance of TiB2@CM-RGD, its in vitro anticancer effect was further evaluated ( Fig.11 Good biocompatibility is the basis for the use of nanomaterials in biomedicine. The cytotoxicity of TiB2@CM-RGD on different cells (U87 human brain astroglioma cells, MCF-7 human breast cancer cells, HaCat human immortalized epidermal cells) was studied using a cell viability assay kit (CCK-8). Fig.11 a is the cell activity of different cells after incubation of different cells with two-dimensional bionic nanomaterial TiB2@CM-RGD for 4 hours at different concentrations (taking the concentration range of 0-200μg / mL as an example). It can be seen that when the concentration of TiB2@CM-RGD is as high as 200μg / mL, the cytotoxicity of the material can be ignored, indicating that the synthesized TiB2@CM-RGD has good biocompatibility.

[0059] In order to detect the generation of ROS in cancer cells and verify the mechanism of TiB2@CM-RGD sonosensitizer to eradicate tumor cells under ultrasound irradiation, the commonly used fluorescent probe 2,7-dichlorofluorescein diacetate (DCFH-DA) staining was performed. The in vitro cell therapy experiment was divided into the following 8 groups: a: PBS group; b: PBS+ultrasound group; c: PBS+photothermal group; d: PBS+ultrasound+photothermal group; e: TiB2@CM-RGD group; f: TiB2@CM-RGD+photothermal group; g: TiB2@CM-RGD+ultrasound group; h: TiB2@CM-RGD+ultrasound+photothermal group. The specific operations are as follows:

[0060] Confocal microscopy was performed on the cells to monitor the production of ROS in different groups and the therapeutic effects after treatment in different groups. U87 cells were first inoculated on 8 cell culture dishes (30 mm in diameter) and incubated for 12 hours. Then, DMEM culture medium containing TiB2@CM-RGD (150 μg / mL) was added to the TiB2@CM-RGD group, TiB2@CM-RGD+ultrasound group, and TiB2@CM-RGD+ultrasound+photothermal group, respectively, and incubated at 37°C and 5% carbon dioxide for 4 hours. The cells were replaced with new culture medium and the experimental group was ultrasounded (40 kHz, 1.5 W / cm 2 ,10min,50%duty cycle) and / or photothermal (808nm,1.0W / cm 2 , 10min), then dyes Hoechst 33342 and DCFH-DA were added to the cell dishes respectively and incubated for 30min, and then imaged using a confocal laser scanning fluorescence microscope.

[0061] like Fig.11c, No obvious green fluorescence was found in groups af. However, obvious green fluorescence was found in groups g and f, indicating that TiB2@CM-RGD can generate a large amount of reactive oxygen species after ultrasound treatment, proving the killing mechanism of sonodynamic therapy.

[0062] Next, the killing performance of TiB2@CM-RGD on U87 cells under the action of laser and / or ultrasound was further evaluated. The specific operation is as follows: For in vitro photothermal and sonodynamic therapy, the standard CCK-8 method was used to detect 8 experimental groups with different treatments. U87 cells were first inoculated on 8 cell culture dishes (30 mm in diameter) and incubated for 12 hours. DMEM culture medium containing TiB2@CM-RGD (150 μg / mL) was added to the TiB2@CM-RGD group, TiB2@CM-RGD+ultrasound group, and TiB2@CM-RGD+ultrasound+photothermal group, respectively, and incubated at 37°C and 5% carbon dioxide for 4 hours. The cells were replaced with new culture medium and the experimental group was ultrasounded (40kHz, 1.5W / cm 2 ,10min,50%duty cycle) and / or photothermal (808nm,1.0W / cm 2 , 10min), then the cells were digested with trypsin, quickly transferred to a 96-well plate, and incubated again for 24h. Finally, CCK-8 was used to test cell viability.

[0063] The cell viability of the above 8 experimental groups was detected using a cell viability detection kit (CCK-8). The relative cell viability of the ae group was greater than 90%, indicating that the use of ultrasound and / or laser irradiation and the TiB2@CM-RGD material of the present invention itself would not cause damage to U87 cells. However, in the case of TiB2@CM-RGD combined with laser irradiation, ultrasound, or laser irradiation + ultrasound, the relative cell viability in the experimental groups fh was 51.2%, 49.3%, and 14.9%, respectively ( Fig.11 b). The results showed that photothermal therapy and sonodynamic therapy alone had certain therapeutic effects, and when the two were combined, the therapeutic effect was significantly enhanced. The combined treatment of photothermal and sonodynamic therapy could produce a significant synergistic effect.

[0064] In addition, the cells treated with different CCK-8 methods were co-stained with calcein-AM / PI (30 min) to detect the live cells (green) and dead cells (red) in different experimental groups, and confocal microscopy imaging was performed. The killing effect of U87 cells after various treatments was shown by live / dead co-staining (AM / PI), which further confirmed the excellent killing effect of combined photothermal and sonodynamic therapy on cancer cells ( Fig.11 d).

[0065] 6. Study on the enrichment effect of two-dimensional bionic nanomaterial TiB2@CM-RGD on brain tumors

[0066] TiB2@CM-RGD was dispersed in water at concentrations of 0, 25, 50, 100, 150, and 200 ppm, and the fluorescence intensity of each solution was measured, as shown in Fig.12 As shown in a, b, it was determined that its concentration was linearly correlated with its fluorescence intensity.

[0067] This example also explored the brain tumor enrichment ability of TiB2@CM-RGD. Mice were used as experimental subjects, and tumor-bearing mice were treated with drugs via tail vein injection at a drug concentration of 10 mg / mL.

[0068] like Fig.12 a, b After confirming that the concentration of TiB2@CM-RGD is linearly correlated with its fluorescence intensity, fluorescence imaging of the brain of BALB / c tumor-bearing nude mice at different time points after intravenous injection of three nanomaterials (TiB2, TiB2@CM, TiB2@CM-RGD) ( Fig.12 c). It can be observed that the fluorescence reached its peak 12 hours after intravenous injection. It is worth mentioning that compared with TiB2 and TiB2@CM, the brain of mice treated with TiB2@CM-RGD showed stronger fluorescence, which proved the significant tumor cell targeting ability of TiB2@CM-RGD.

[0069] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and changes without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A two-dimensional biomimetic titanium diboride nanomaterial for photothermal and / or sonodynamic therapy of tumors, characterized in that The two-dimensional bionic titanium diboride nanomaterial is obtained by coating titanium diboride nanosheets with cell membranes and modifying them with phospholipid-polyethylene glycol-RGD; the size of the titanium diboride nanosheets is 50-100 nm and the thickness is 0.5-2 nm; and the cell membrane is a J774A.1 macrophage membrane.

2. The two-dimensional bionic titanium diboride nanomaterial according to claim 1, characterized in that The titanium diboride nanosheets are prepared by a liquid phase exfoliation method, comprising the following steps: S1. Evenly dispersing titanium diboride powder in water to obtain a titanium diboride aqueous solution with a concentration of 0.1-1 mg / mL; the titanium diboride powder is micron-sized titanium diboride block particles; S2. Place the titanium diboride aqueous solution obtained in S1 into a microwave synthesizer for microwave synthesis. The supernatant obtained by centrifugation after the reaction is the titanium diboride nanosheet dispersion; wherein the microwave synthesis temperature is 80-110 °C, the power is 10-50 W, and the time is 10-30 min.

3. The method for preparing the two-dimensional bionic titanium diboride nanomaterial according to claim 1, characterized in that The following steps are involved: (1) Preparation of titanium diboride nanosheet dispersion by liquid phase exfoliation method; (2) mixing the titanium diboride nanosheet dispersion with the cell membrane and performing ultrasound, centrifuging the mixture after ultrasound to remove excess cell membrane in the supernatant, and redispersing the resulting precipitate in deionized water to obtain a cell membrane-coated titanium diboride nanosheet dispersion; (3) The cell membrane-coated titanium diboride nanosheet dispersion is mixed with phospholipid-polyethylene glycol-RGD in a certain mass ratio, and reacted in a constant temperature oscillator for a period of time to obtain a two-dimensional bionic titanium diboride nanomaterial.

4. The method for preparing the two-dimensional bionic titanium diboride nanomaterial according to claim 3, characterized in that In step (1), the concentration of the titanium diboride nanosheet dispersion is 0.05-0.15 mg / mL.

5. The method for preparing the two-dimensional bionic titanium diboride nanomaterial according to claim 3, characterized in that In step (2), the mass ratio of titanium diboride nanosheets to cell membranes is 4:1-2:

1.

6. The method for preparing the two-dimensional bionic titanium diboride nanomaterial according to claim 3, characterized in that In step (3), the mass ratio of the cell membrane-coated titanium diboride nanosheets to the phospholipid-polyethylene glycol-RGD is 1:1-3:

1.

7. The method for preparing the two-dimensional bionic titanium diboride nanomaterial according to claim 3, characterized in that In step (2), the cell membrane is obtained by extracting the cell membrane using a kit after cell lysis.

8. Use of the two-dimensional bionic titanium diboride nanomaterial according to claim 1 as an agent for preparing photothermal and / or sonodynamic tumor therapy.

9. The use according to claim 8, characterized in that Ultrasound conditions for sonodynamic therapy are: power 1-2.5 w / cm 2 , ultrasound time is 1-10 min; the laser conditions for photothermal therapy are: wavelength 808~1064 nm, direct irradiation time is 1-10 min.

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